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263 Cards in this Set
- Front
- Back
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what makes RNA more susceptible to degradation than DNA?
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The 2' OH group.
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what happens to uracil in DNA?
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Deamination of cytosine to uracil is potentially mutagenic so Uracil bases in DNA are repaired
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nucleoside
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a base bonded to a sugar (minus the phosphate)
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nucleotide
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a nucleoside bonded to phosphate groups (by ester linkages)
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secondary structures of DNA
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hairpins: short turn with 6-8 nucleotides
stem loops: single stranded loop with hundreds of nucleotides |
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tertiary structures of DNA
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pseudoknot - (like a figure eight)
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how are three dimensional DNA structures formed
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by hydrogen bonds
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ribozymes
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RNA molecules that catalyze reactions (the proof of the RNA world hypothesis)
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ribopolymerase
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a ribozyme that is an RNA-directed RNA polymerase
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retrovirus
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a virus containing RNA and reverse transcriptase. the RNA gets converted to cDNA in the host cell and integrated into the genome for stability.
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three basic RNA types in bacteria
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rRNA (80%)
tRNA (15%) mRNA (5%) |
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shine delgarno sequence
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a sequence of RNA that base-pairs with rRNA.
known to be purine-rich (A/G) it is located upstream of the AUG initiation codon that codes for the fMet residue. |
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polycistronic
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characteristic of most bacterial mRNAs.
it means that one mRNA can code for multiple proteins. Intervening sequences between coding sequences serve as docking sites for ribosomes (shine delgarno sequences |
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what intitiates translation in eukaryotic mRNA?
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the presence of the first AUG. it is recognized by the tRNA and then the ribosomes are brought over.
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stop codons
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UAA, UAG, UGA
these are read by specific release factors which causes the ribosomes to release the protein |
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release factors
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proteins that recognize stop codons and cause the release of the protein from the ribosome.
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how to detect introns by electron microscopy
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hybridize an mRNA to genomic DNA. if there is one loop, then the DNA sequence is continuous.
if there are two loops (or more) then the gene contains an intron (or more) |
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prokaryotic ribosome structure
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70S --> 50S + 30S
50S --> 23S + 5S + 31 proteins 30S --> 16S + 21 proteins |
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eukaryotic ribosome structure
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80S --> 60S + 40S
60S --> 28S + 5.8S + 5S + 50 proteins 40S --> 18S + 33 proteins |
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tRNA structure
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phosphorylated 5' terminus
amino-acid attachment site (-OH on an CCA) at the 3' terminus cloverleaf pattern anti-codon loop near the center half of the molecule is base-pairing 4 dS regions stack to form an L 3D shape. |
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aminoacyl tRNA
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a tRNA that is bonded with a residue.
the residue is esterified to the 2' or 3' OH of the terminal A of the tRNA |
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main difference between transcription/translation in prokaryotes and eukaryotes
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prokaryotes: can take place simulatenously (no compartmentalization
eukaryotes: take place in different places so are spatially and temporally separated |
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other RNA that is important in protein synthesis in eukaryotes
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snRNA: participate in splicing
microRNA: small mRNA that hybridize and inhibit the translation of complementary mRNA siRNA: (small interfering RNA) can silence mRNA through similar mechanisms as miRNA snRNP: ribonucleoproteins (i.e. ribozymes) |
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how to label the 2 strands of DNA
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template strand (the strand that is complentary to the RNA)
coding strand (the strand that is identical to the RNA...except for the T to U) |
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what subunit of RNA polymerase (in bacteria) recognizes the promoter?
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the sigma site (σ)
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the subunits of RNA polymerase in bacteria
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alpha (2 identical subunits)
beta (2 dissimilar subunits) sigma |
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highly conserved promoter sequence in prokaryotes:
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at -35: TTGACA
at -10: TATAAT (Pribnow box) |
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highly conserved promoter sequence in eukaryotes:
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at -25: TATAA (TATA box)
sometimes there is a CAAT box at -75. there are also enhancers that can enhance the binding ability of the polymerase |
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different sigma subunits in RNA polymerase
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sigma 70: the default subunit
sigma 32: synthesized when the cell is starving, it specifically binds to genes that synthesize heat shock proteins (different promoter sequences) |
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the active site of RNA Polymerase
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similar to DNA polymerase:
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what types of modifications are done to mRNA in prokaryotes?
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NONE!!
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what types of modifications are done to mRNA in eukaryotes
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5' cap
3' polyA tail splicing editing (deamination of cytidine) |
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how does RNA polymerase attach to the DNA?
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the sigma subunit runs along the DNA until it reaches the -35, -10 promoter sequences.
then it recruits the other subunits to start transcription. |
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rna polymerase active site (prokaryotes)
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there are 2 metal ions (Mn or Mg). One attaches to the enzyme and the other comes attached to the nucleoside
the main amino acid is aspartate. The 3’ hydroxyl group of the growing chain attacks the alpha phosphoryl group of the incoming nucleoside triphosphate. |
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de Novo
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means without primers.
in transcription, the RNA polymerase works without primers. |
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what is found at the 5' end of RNA (before modifications)
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a triphosphate group
the first RNA base is usually a purine |
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what is the reaction that RNA polymerase catalyzes:
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(RNA)n + NTP --> (RNA)n+1 + PPi
requires NTP and metal ions |
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what can RNA polymerase do other then catalyze the addition of nucleotides?
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it unwinds DNA (about 17bp) and rewinds it in the rear.
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transcriptional stop signal
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the simplest version is a palindromic GC rich region followed by an AT rich region containing 4 Uracil residues
Polymerase pauses after synthesizing a hairpin – the U-A base pairs are weak – causing the nascent RNA to dissociate the Rho signal (without it, the RNA would be much longer) |
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mechanism of Rho termination
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Rho binds to C rich region of ssRNA and hydrolyzes ATP to dissociate the RNA
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rifampicin
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antibiotic that inhibits transcription by binding with the polymerase
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actinomycin
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antibiotic that inhibits transcription by binding tightly and specifically to double helical DNA preventing it from being an effective template for RNA synthesis.
can inhibit RNA transcription while retaining DNA synthesis! |
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how are rRNA and tRNA processed in prokaryotes
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they are transcribed as one long transcript and then cleaved and modified.
rRNA: RNAase III cleaves the 5S, 16S and 23S tRNA: RNAse P generates the correct 5' end other enzymes add CCA to the 3' end and modify bases |
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how can nucleotides of RNA be modified?
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can be methylated or isomerized
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how many eukaryotic RNA polymerases?
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3. Pol I, II, and III
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RNA Pol I
what does it encode? TF's? Location? Regulated? |
synthesizes rRNA (except 5S)
located in nucleolus has 2 TF's not very regulated rapid continous transcription |
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Pol II
what does it encode? TF's? Location? Regulated? |
synthesizes mRNA and snRNA's
located in nucleus many TF's inhibitors and enhancers highly regulated transcription only occurs when proper proteins are bound to the promoter |
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Pol III
what does it encode? TF's? Location? Regulated? |
encodes tRNA and 5S rRNA
located in nucleus has 3 TF's: TFIIIA, TFIIIB, TFIIIC Regulated by DNA sequences within the transcribed region no promoter region needed |
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what is common about all RNA polymerases in eukaryotes
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TF's are needed in addition to RNA Pol for transcription to take place
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alpha amanitin
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poisonous mushroom has effects on RNA Pol:
I: nothing II: inhibited by low concentrations III: inhibited by high concentrations |
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CTD
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carboxy terminal domain.
found at the 5' end of RNA Pol II is inactive when not phosphorylated (i.e. during the preinitiation complex) can be phosphorylated by TFIIH which starts elongation also plays a major role in post-transcriptional modifications (recruiting the necessary proteins) |
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similarities between eukaryotic and prokaryotic RNA Polymerases
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eukaryotic has Beta like subunits and Alpha like subunits. but they then have many additional subunits
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omega subunit
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it is an essential subunit in eukaryotic RNA polymerase activity. however in prokaryotes, it is now considered part of the core for the polymerase but it is not essential for transcription. Rather it stabilizes enzyme and assists in assembly of subunits
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Pol I transcription factors
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upstream activator element (UAF) binds to UCE (upstream control element) of DNA. it is located at -100. it also binds to the core element directly upstream from the transcriptional start site
then it recruits selectivity factor I complex (SFI and TIF-IB) which then recruits the polymerase for transcription |
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Pol III Transcription factors
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TFIIIC binds to the two control elements upstream of the transcriptional start site
it then recruits TFIIIB to bind to sequence 30 bp upstream from TSS TFIIIB has TBP (interact w/ DNA) and BRF (interacts with Pol III) TFIIIB then recruits Pol III for transcription TFIIIA is only used for 5S transcription. it binds to DNA using zinc-fingers. it then recruits TFIIIC and then follows the normal transcription route |
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Pol II promoter/enhancer sites
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TATA box and Inr.
housekeeping genes have neither and are transcribed at very low constant levels. to get the highest level of transcription, should have both TATA and Inr Dpe: downstream enhancer |
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Pol II Transcription Factors
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TFIID (contains TBP and TAF's) bind to DNA at the TATA, Inr and Dpe sequences
then TFIIB interacts with C-terminus of TBP and DNA. it then recruits TFIIF which is associated with Pol II. Pol II comes with TFIIF, TFIIE, TFIIH, and SRB TFIIE activates TFIIH TFIIH has helicase activity, kinase (for CTD), and DNA repair and cyclin dependent activity. SRB remodels chromatin, activates splicing, is connected to RNA Pol II through the CTD |
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TBP
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TATA binding protein
binds TATA with high affinity and interacts with the minor groove and bends the DNA. it interacts with TBP associated factors (TAF's) for modulation C-terminus is highly conserved and N-terminus is not but both are neccessary |
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how does eukaryotic transcription occur after the preinitiation complex has completed
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TFIIH hydrolyzes ATP and phosphodiester bond is formed.
then TFIIH phosphorylates CTD then ELONGATION. then TFIIB and TFIIE leave to dissociated RNA Pol II, CTD needs to be dephosphorylated. |
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6 steps in protein regulation
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1) transcriptional control (***KEY***)
2) RNA processing control 3) RNA transport/localization control 4) translational control 5) mRNA degredation control 6) protein activity control |
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trans activators and cis regulators
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proteins that bind to DNA sequences far away from the gene (or very close (cis)) but can help stablize the preinitiation complex and activate transcription
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cis-acting elements regulatory role in transcription
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- some are necessary for basal activity of gene (TATA box)
- some are designed for gene activation in response to changes in environment - some are meant to be activated in specific tissues (tissue specific elements can bind and increase transcriptional rate) |
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how do DNA binding proteins interact with DNA?
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most use the major groove because it has more connections available
- zinc finger (c2h2, c4, or c6) - leucine zipper - helix-loop-helix - homeobox |
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transcriptional co-activators
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proteins that bind to sequences that are very far away from the transcriptional site can still influence transcription by DNA looping and can interact with TF's and influence their activity and affinity
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how to determine if a protein binds to a region of DNA
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DNA footprinting
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zinc finger
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c2h2: 3 fingers
c4, or c6 each finger can contact three nucleotides in the major groove through alpha helix can form heterodimers and inhibit |
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homeobox
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popular by the fly having a leg in its face!
one helix (60 aa) fits in major groove of DNA. highly conserved |
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leucine zipper
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2 domains dimerized. basic amino acids bind to the DNA.
The dimerization domain contains leucine or other hydrophobic amino acids every seventh amino acid which form hydrophobic bonds between monomer helices - coiled coil. |
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helix-loop-helix
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Helix-loop-helix motif has an N-terminal basic helix, a middle loop region, and a C-terminal region with hydrophobic amino acids spaced at intervals characteristic of an amphipathic alpha helix.
kind of like leucine zipper can form heterodimers and inhibit! |
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use of heterodimers
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expands the number of potential dna binding sequences.
they can only be formed between members of the same class!! |
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what is unusual about the 5' cap
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usually, the 5' P attacks the 3' OH on the ribose.
here the 5' P (diphosphate) attaches to the alpha 5' P of the GTP and so there is a 5'-5' linkage as opposed to 5'-3' linkage N-7 of G is methylated in all caps |
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difference between cap 0, 1, 2
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cap 0: only N-7 is methylated
cap 1: N-7 and the first ribose (past GTP) cap 2: N-7 and the first two riboses (past GTP) |
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tRNA modifications
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- chemically modified bases
- 3' end cleaved and CCA added - splicing (sometimes in euk) - 5' end cleaved |
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rRNA modifications
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- cleavage
- nucleotide modifications - 5' splicing |
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5' cap's role
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stabilizes mRNA
enhances translation |
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role of polyA tail
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NOT needed for transport
enhances translation **KEY stabilizes mRNA |
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how is polyA tail added
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the termination sequence of RNA is A rich and recognized by an endonuclease which cleaves it and another enzyme adds 250 or so A using ATP!
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how can you have two identical pre-mRNA's encoding proteins of different lengths?
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alternative splicing...
OR cytidine deamination (CAA-gln UAA-STOP!) |
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typical intron sequence:
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upstream exon AG GUAAGU..........Branch site/A...........(Pyrimidine)10 NCAG G downstream exon
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how does intron begin and end
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begins with GU and ends with AG
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what is upstream exon sequence, branching sequence, and downstream exon sequence in splicing
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usptream: AG
branch: A downstream: G |
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spliceosome
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A large complex of proteins and small RNA responsible for splicing.
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spliceosome reaction
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1. Cleavage of phosphodiester bond between exon 1 and
the 5’ end of the intron The attacking group is the 2’-OH group of an adenylate residue in the branch site. 2. A 2’ - 5’ phosphodiester bond is formed between A and 5’ terminal phosphate of the intron (transesterification). this results in the release of exon 1 3. The 3’-OH terminus of exon 1 attacks the phosphodiester bond between the intron and exon 2, to join exon 1 and 2 and the intron is released. |
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snRNP
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small nuclear ribonucleoproteins
they are RNA's in complex with proteins |
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alternate splicing
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in a gene with X introns (or X+1 exons) the number of possible mRNAs due to alternate splicing are
2 ^ X |
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what is the 3' RNA sequence that signals the endonuclease to cleave and then attach a polyA tail?
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AAUAAA
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rRNA splicing
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can be self-spliced. the introns are called group I introns.
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rRNA processing
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Pol I --> 45S
45S --> 28S, 18S, 5.8S 18S + proteins --> 40S 28S + 5.8S + proteins --> 60S |
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what enzymes process rRNA
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sno RNPs modify ribose and base components in the rRNA (e.g. aiding methyl groups and changing uridine to
pseudouridine) nucleases cleave the large precursor to the three mature RNA's. |
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tRNA processing
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1. The 5’ leader is cleaved by RNAse P
2. The 3’ trailer is removed and CCA is added by CCA adding enzyme 3. Ribose and bases are modified (as the rRNA) 4. Many eukaryotic pre-tRNA are spliced by an endonuclease and a ligase to remove introns 1 in every 10 bases are modified |
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β-galactosidase:
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enzyme that converts lactose into glucose and galactose.
there are small amounts of allolactose (glucose+galactose in a 1,6 bond) encoded by Lac Z |
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Galactoside permease:
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protein that transports lactose across the bacterial membrane
encoded by Lac Y |
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Thiogalactoside transacetylase:
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detoxifies compounds that are brought in by permease
encoded by Lac A |
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operon
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A transcription unit in prokaryotes that consists of regulatory regions and several genes in a cluster.
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basic fundamental functions of the lac operon in the presence/absence of lactose
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in the presence of lactose, the
genes Z, Y, A are activated. In medium containing glucose the activity is turned off within minutes. |
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how can one follow beta-galactosidase activity?
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IPTG: is a small molecule that resembles lactose and can greatly induce the expression of beta-galactosidase
also, X-GAL is transported by lactose permease and hydrolyzed by β-galactosidase, producing a blue product which gives the colony a blue color. |
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2 types of regulatory proteins in bacteria
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- positive acting proteins
- negative acting proteins: Negative-acting proteins are called Repressors, and the DNA site to which they bind is call Operator. The operator sequence is always close to the promoter sequence. |
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repressors
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they are negative acting proteins that bind to an operator which is always close to the promoter sequence.
they bind to effectors Effectors can be inducers Effectors can also be Corepressors to activate the repressor |
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effector
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compound that can bind to a repressor and either activate it or deactivate it.
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in absence of lactose, how does the lac operon get affected?
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the repressor mRNA is transcribed using the first promoter.
The repressor protein binds to the operator and prevents transcription of the downstream promoter |
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what happens to lac operon when lactose is present
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lactose acts as an effector (inducer) to repress the repressing activity of the repressor.
it is a natural inducer. thus the Lac A, Y, Z can be expressed |
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cAMP and the lac operon
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in the absence of glucose, cAMP levels INCREASE
high cAMP levels are a signal for starvation and induce beta galactosidase expression but cAMP alone is NOT enough. need CRP (cAMP Receptor Protein aka CAP) cAMP-CAP and RNA polymerase bind to each other weakly in the absence of DNA. By itself, RNA polymerase binds fairly weakly to the lac promoter. However, when cAMP-CAP and RNA polymerase bind to the lac operon together, they stimulate each other’s binding |
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dibutyryl cAMP
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acts as a transcriptional activator for the lac operon.
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cooperative binding
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situation where two proteins bind with a greater affinity to a substrate when they are both present in solution (i.e. cAMP-CAP and RNA Pol)
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two lines of defense of the human body
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innate immune system: responds rapidly to features present in many pathogens
adaptive immune system: responds to specific features in a given pathogen |
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how does the immune system work?
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identifies the features on disease-causing organisms and then works to eliminate them.
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specificity and self tolerance (immune system)
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specificity: recognizing foreign antigens
self-tolerance: destroying them without harming the host |
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examples of the innate immune response
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monocytes, macrophages, dendritic cells, natural killer cells, granulocytes
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examples of the adaptive immune response
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B lymphocytes
T lymphocytes |
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two lines of defense of the human body
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innate immune system: responds rapidly to features present in many pathogens
adaptive immune system: responds to specific features in a given pathogen |
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how does the immune system work?
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identifies the features on disease-causing organisms and then works to eliminate them.
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specificity and self tolerance (immune system)
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specificity: recognizing foreign antigens
self-tolerance: destroying them without harming the host |
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examples of the innate immune response
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monocytes, macrophages, dendritic cells, natural killer cells, granulocytes
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examples of the adaptive immune response
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B lymphocytes
T lymphocytes |
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immunodeficiency
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the breakdown of the immune systems ability for specificity
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autoimmune
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break down of self-tolerance
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what is the rate limiting step in protein folding?
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from secondary to tertiary structure
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domains of hsp 70
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ATPase domain, peptide binding domain, and a cap
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atp binding region of hsp 70
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highly conserved region.
requires Mg and K |
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peptide binding domain of hsp 70
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highly variable
binds short hydrophobic regions |
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dnaK
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another name for hsp70 in bacteria
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why are the experiments done on protein refolding different than the real deal?
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in the real deal, the protein is coming out slowly from the ribosomes, and you want to make sure that no unwanted folding takes place until the entire protein is out.
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what happens if the protein does not fold correctly
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hsp 70 recognizes this by the hydrophobic regions still sticking out, so hsp70 rebinds and then gives the protein another chance!
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hsp70 bound to adp vs atp
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when bound to adp: the latch is closed and the polypeptide cannot break free
when bound to atp, the latch is open and the polypeptide can break free |
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DNA J
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peptide that works with hsp 70. it binds to the DNA and recruits the hsp70 and promotes atp hydrolysis to latch onto the protein
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peptidyl-prolyl isomerase
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enzyme that sits at the end of the ribosome to isomerize certain residues before hsp70 latches to it.
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what does hsp70 stand for? why?
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heat shock protein.
because it protects proteins during heat shock. it binds to the denatured proteins to prevent unwanted interactions between denatured proteins. |
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hsp 10
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the cap for the hsp 60 chaperone
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how does hsp60 work?
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it has 7 subunits. inside there are hydrophobic regions
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how is hsp60 different from hsp 70
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it doesn’t bind to COMPLETELY unfolded protein. It likes preformed secondary structures unlike hsp70.
also it completely encases the protein unlike the hsp 70 |
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aggregates
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form when misfolded proteins accumulate.
small aggregates are far worse then large ones. they form plaques. many cells have machinery to prevent the aggregates (except animals) |
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how to get rid of aggregates?
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use hsp 104 (looks like hsp60)
has slicers on its side to pick up and carry away the small misfolded proteins binds atp. when it binds, the two rings grind against one another in animals the aggregates are brought using microtubule/dynein to the centrosomal area and can harmlessly accumulate there |
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what is the difference between cytosolic and non-cytosolic environment
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cytosolic: oxidizing environment (i.e. bad for cys and met)
non-cytosolic: reducing environment |
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how to measure the rate of degradation of a cell?
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flood a cell with radioactive S and then measure the rate of excretion (this is the rate of degradation)
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what is the probable function of short-lived proteins
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rapid response
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what is the probable function of long-lived proteins
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a protein that should remain at constant levels.
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Jun protein function
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a transcription factor for stress response
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major differences between cellular digestive and organism digestion?
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specificty, processivity, and energy consumption
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specificty (digestion)
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stomach: none! want to chew everything up!
cell: YES!! must be very specific so that the cell isnt eaten! |
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processivity (digestion)
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stomach: doesnt have to be completely processed, there could be fragments.
cell: must be completely broken down into aa and other molecules |
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energy consumption: cell v gut
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stomach: no energy used
cell: uses energy |
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clpP
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degradation protein in bacteria
has a chamber (like hsp proteins) but has scissors inside to cut the protein. this is all energy independent. the energy is needed to selectively bring the protein in! |
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clpA
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part of protein degradation in bacteria.
sits on top of clpP. responsible for bringing in the proteins that need to be degraded. it is like a chaperone that recognizes the protein, and unfolds it and sends it into clpP. there are a variety of homologs of clpA that recognize different signals for degradation |
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Lon protease
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very large protease.
pretty much a combination of clpA and P. has both chambers for bringing proteins in and digesting them. could be an evolutionary precursor to regular chaperones since if a mutation occurs in the protealytic site, than it would behave much like hsp60! |
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2 sites of degradation in eukaryotes
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proteasome and lysosome
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which proteins are sent to proteasome
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-short lived regulatory proteins
-small amount of long-lived proteins -abnormal proteins |
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which proteins are sent to lysosomes
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-most long-lived proteins
-membrane proteins -aggregated proteins |
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ubiquitin
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a polypeptide shaped like a noose
it marks substrate proteins for degradation. To be delivered to proteasome, it needs to be covalently attached to multiple ubiquitins |
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most important residues on ubiquitin
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glycine residue: to attach to cys of E1(c terminus)
lysine residue: to attach to the next ubiquitin |
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sequence of events to ubiquitination
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E1 binds to U.
then it transfers U to E2. E3 meanwhile, binds to the protein and then E2 finds E3 and transfers the U to the protein--attaches to lysine on the protein |
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how many E1, E2 and E3's are there and why?
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E1: 1
E2: a few E3: thousands the reason is because you only need one E1 and a few E2's to recognize the thousands of E3's which need to vary so that it can bind to different domains on degradable proteins |
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structure of proteasome
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4 subunits. in bacteria they are all the same but in eukaryotes they can be different.
the beta chambers in the middle are for degradation. but the protein must be denatured first. another peptide (analogous to clpA) recognizes the ubiquitin markers and binds the protein and denatures it. it then sends it to the 20S proteasome for digesiton. |
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functions of the lid (19S structure that assists the proteasome)
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recognize the 4 ubiquitin chain.
recognize substrate and transfer to the base isopeptidase (destroy the peptide bond between the ubiquitin branches) release the free ubiquitin. |
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how does proteasome chew a protein?
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like a dog. first cuts into large pieces then smaller.
the first bite is with trypsin (arg, lys) |
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HDAC 6
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histone deacetylation complex
doesnt actually do that since it is in the cytosol. binds to ubiquitin and sends it to dynein to carry off to the aggresome. |
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how can ligands affect expression?
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Protein-protein interactions occur between DNA binding proteins and co-activators (i.e. the ligands) to regulate transcription.
sometimes the ligand can cause the release of a repressor or activation of an enhancer, or the opposite. |
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glucocorticoid receptor and how it works with nuclear localization
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wihtout the ligand, there is a repressor bound to the protein.
when the ligand binds, the repressor leaves and exposes the NLS. so the complex goes to the nucleus and regulates transcription |
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what happens after chromatin is activated
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transcription factors can bind, and cis-acting proteins, mediators, co-activators...
transcription begins! |
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histones
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proteins that help bundle up DNA into inactive chromatin. they interact with DNA through the minor groove
4 different kinds. H1-H4 H1 is the most unique, it forms as a linker between nucleosomes |
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dna interaction with histones
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histones bind at the minor groove.
DNA surrounds the 4 core histones with an amino tail protruding out. this tail is important in docking proteins |
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HAT
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histone acetyl transferase.
acetylates histones which activates the chormatin to allow for transcription. does so by transferring acetyl from acetyl CoA to the lysine of the amino tail of the histones which decreases affinity for DNA and increases affinity for bromodomains which are characteristic of many TF's and other DNA binding proteins |
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TFIID and histones
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TFIID contains a bromodomain and an acetyl transferase.
so it is capable of activating chromatin and recruiting Pol II |
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2 ways of activating chromatin
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- acetylation
- chromatin remodeling (must be acetylated first before recruitng the remodeling engine) |
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2 ways of deactivating chromatin
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- deacetylation
- remodeling |
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epitope
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region of the antigen that binds to the antibody
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how does epitope interact with antibody?
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non covalent interactions
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where are t and b cells synthesized?
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in the central lymphoid organs: bone marrow
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where are t cells maturized
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in the thymus
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where do t and b cells go after syntehsis
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to the spleen and other peripheral lymphoid organs and filter the blood for antigens
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constant regions of the heavy chain?
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responsible for antibody-antibody interactions such as dimerization or pentamerization...
hold the chains together also, carbohydrates are present to stimulate dimerization and phage recognition |
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variable regions of the chains
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responsible for binding the antigen
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secondary structure motif of b cells
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beta sandwich. many similar antiparellel beta sheets with loops that are variable.
these loops are where the epitope binding occurs |
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CDR
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the loops of the secondary structure of the b cell.
these are responsible for the variety in b cells |
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avidity vs affinity
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avidity: the strength of an antigen binding to the antibody on both of the variable chains.
affinity: the strength of an antigen binding to the antiboy on one chain avidity is hundreds times stronger than affinity |
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Fab
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the two slants of the Y of the b cell.
THese are the parts of the b cell that have the binding to the antigen |
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Fc
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the stem of the Y of the b cell
mostly repsoible for dimerization and such |
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IgA
-secreted form -subclass -H chain -L chain |
dimerized with J
has IgA1 and IgA2 H chain has alpha1 or alpha2 L chain has kappa or lambda |
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IgM
-secreted form -subclass -H chain -L chain |
penatmeric with J
none H chain has Mu L chain is kappa or lamnda |
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IgD
-secreted form -subclass -H chain -L chain |
monomeric
none H chain has delta L chain has kappa or lamda |
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IgG
-secreted form -subclass -H chain -L chain |
monomeric
none IgG1-4 H chain has gamma1-4 L chain has kappa or lamda |
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IgE
-secreted form -subclass -H chain -L chain |
monomeric
none H chain is epsilon L chain is kappa or lamda |
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different Ig isotypes
what determines them? |
IgA, IgD, IgE, IgG, IgM
the amino acid sequence of the CH chain determines which! |
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B cell differentiation to antibody secretion
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as B cells proliferate, they become B blasts with less memrbane bound antibodies
and then eventually become plasma cells which secrete IgM class first! |
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somatic hypermutation
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when a B cell gets activated by an antigen, it proliferates and an enzyme AID (activation induced deaminase) helps the activated B cell hypermutate in the hopes that one mutation will develop a greater avidity/affinity for the antigen and will become the new primary antibody
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AID
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activation induced deaminase
an enzyme that helps b cells hypermutate by deaminating cytidine to cause transition mutations |
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how many classes of antibodies can a single b cell release
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1
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how many antibodies are present in an immune response
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many
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which class of antibodies are responsible for the primary response?
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IgM
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which antibodies are responsible for the secondary response
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IgG, IgA, IgE. and they have greater affinity so this makes the secondary response greater.
example of memory |
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t cell receptor
2 different types |
2 chains with globular domains.
V and C regions contain CDR's 1) alpha-beta 2)gamma-delta |
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t cells
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they do not secrete anything, rather they bind to the antigen and either kill it themselves or recruit other molecules to kill it
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how do t cells work?
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each TCR is capable of recognizing foreign proteins that are held in the MHC (major histocompatibility complex).
they then bind to it for an immune response |
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MHC
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major histocompatibility complex, brings samples of intracellular proteins out of the cell and holds them in place outside the cell for recognition
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which receptors have a VJ chain
which have a VDJ chain |
VJ: B--> L chains (kappa and lamda)
T: alpha and gamma VDJ: B-->H chains T: beta and delta |
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how does the C region go?
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mu-delta-gamma3-gamma1-alpha1-gamma2-gamma4-epsilon-alpha2
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how does rearrangement go at the site of the DNA locus
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All the genes upstream and downstream of the rearranging genes remain in the locus
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sources of diversity in T and B cells
(9 total) |
combinatorial: alignment, cleavage, and religation
inexact joining: deletion/insertion, extra D or no D region (T only) or reading of D in all three regions (T only) random chain association in TCR's (1000 alpha x 1000 beta = 1,000,000 possible TCR) class switching (B only) somatic hypermutation (B only) |
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where does somatic hypermutation and class switching occur?
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peripheral lymph organs
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peripheral lymph organs
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spleen, lymph, tonsils, peyers patches
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where are switch regions located in b cell blasts?
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after every constant region except in between mu and delta
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DNA locus of rearrangments
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contains all the V regions up to the V of choice.
contains only the D region of choice (if it has a D region) contains the J region of choice and all J's after (they eventually get spliced) contains the C region of choice and all C's after that |
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Primary RNA transcript of recombination vs mature RNA
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primary: only contains the V of choice. only contains the D of choice, contains the J of choice and all J's after, contains the C of choice and all C's after
mature: only contains the V of choice, only contains the D of choice, only contains the J of choice, and only contains the C of choice. |
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3 stop codons
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UAA, UAG, and UGA
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wobble
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explains how one tRNA can recognize multiple codons.
the third codon doesn't bind well with the tRNA because it is very far away and the two compounds react in a strict orientation, so it is mostly irrelevant |
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how can nucleotides interact in a wobble?
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in the anti-codons:
G--> C or U C--> G A--> U U--> A or G I--> A, U or C |
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reaction of aminoacylation:
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ATP + amino acid --> AMP-AA + PPi
AMP-AA + tRNA --> AA-tRNA + AMP tRNA binds to AA at the 3'OH |
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mechanism of amino-acylation reaction
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one enzyme does everything. the 2 step process increases the fidelity of the reaction
the enzyme binds an aa and makes aminoacyl adenate. there is specificity in recognition After that, the enzyme binds tRNA and this leads to conformational changes in enzyme. Enzyme pauses for a little and decides whether this particular aa corresponds to this particular tRNA. If it understands that it’s a match, it makes the finished product. |
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why would aggregate form in purkinje cells more often then other cells?
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bc purkinje cells do not divide and so the aggregates are not dilluted.
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prokaryotic ribosomes
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70S --> 50S and 30S
50S --> 23S+5S+34 proteins 30S --> 16S + 21 proteins the RNA are the catalytic parts. the proteins are there for structure. in fact, if there were no proteins, the ribosome would still assemble |
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where does the mRNA attach to the ribosome? (pro)
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30S subunit
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3 steps of translation in pro karyotes?
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initiation, elongation, termination
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initiation of translation in prokaryotes
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30S subunits slides upstream until it reaches AUG (met) next to the shine delgarno sequence that binds with the 16S rRNA.
special initiator tRNA is used for the first aa. IF2 binds GTP+fMet+tRNAf (cannot recognize any other tRNA) IF1,3 bring in mRNA and 30S complex then 50S joins and IF1,2,3 leave and GTP is hydrolyzed the formation of 70S marks the end of initiation |
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3 spots in the 50S subunit of ribosome (prokaryotes)
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A(aminoacyl), P(peptidyl), E(exit)
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elongation in prokaryotes translation
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Ef-Tu binds with aa-tRNA and GTP and brings it to the A spot
(Tu interacts with any tRNA except tRNAf) if the tRNA matches with the codon of the mRNA in the A spot, then GTP will hydrolyze and "click" if not, the aa-tRNA will dissociate. the GTP hydrolysis causes GDP+EF-Tu to leave and tRNA-aa stays in the A spot. then the peptide in P spot attacks the aa in A spot. (transpeptidation) Now there is an empty tRNA in P spot and peptidyl-tRNA in A spot. Ef-G with GTP binds to the A spot of the ribsome, hydrolyzes GTP and makes the mRNA (with attached tRNA's) move one spot over in the 5' direction. (translocation) tRNA in E spot leaves, and there is an A spot open and P spot has peptidyl-tRNA |
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what reactivates EF-Tu+GDP?
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Ef-Ts
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translocation (prokaryotes)
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Ef-G binds and hydrolyzes GTP to make mRNA shift one spot over and cause all the tRNAs to shift one spot over.
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termination (prokaryotes)
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Rf1: UAA, UAG
Rf2: UAA, UGA when stop codon appears, the Rf attaches instead of tRNA recruits Rf3 which causes ribosomes to detach. |
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Streptomyocin
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antibiotic: in low concentrations causes ribosomes to make a lot of mistakes.
it interferes with binding of aa-tRNA into the A site. If you increase the concentration, it will stop the binding of tRNAf and block translation |
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Tetracyclin
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antibiotic: inhibits binding to A site
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Erythromycoin
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antibiotic: binds to 23S rRNA and specifically inhibits translocation step.
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Chloramphenicol
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antibiotic: inhibits peptidyl transferase on 50S subunit.
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Puromycin
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antibiotic: mimics aa-tRNA and can get transiently into A site instead of aa-tRNA. That ribosome will put the growing peptide on puromycin instead of aa-tRNA. But association of puromycin is transient and weak and readily dissociates with the bound nascent peptide!! If you add puromycin, you will have formation of a lot of premature terminations
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important differences between prokaryotic and eukaryotic translation
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1) euk takes place in different compartment so there is more added levels of regulation. while in pro, translation takes place during transcription so there is no regulation.
2) euk mRNAs have longer half-lives and are more stable 3)euk are always monocistronic and pro are always polycistronic 4) euk mRNA is modified, pro does have a polyA tail but it specifically signals for degradation! 3) |
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mature mRNA in eukaryotes have:
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5' cap
3' polyA tail 5' UTR with the kozak sequence 3' UTR, ORF (starting with AUG) |
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kozak sequence, how is it different from shine delgarno?
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sequence in 5' utr that signals ribosomes in eukaryotes.
A/GCCAC different because it has no homology to the rRNA (unlike SD and 16S rRNA) rather it increases affinity for transcription factors |
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eukaryotic ribosomes
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80S --> 60S and 40S
60S --> 28S, 5.8S, and 5S + 50 proteins 40S --> 18S and 30 proteins |
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eiF-6
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maintains equilibiurm between 80S and 60,40S by inhibiting the interactions between 60,40S subunits
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translation pre-initiation of mRNA in eukaryotes
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The 5' cap structure is recognized by eIF-4E.
4G (scaffold protien) then binds to 4E and mRNA. 4G then recruits 4A which has RNA helicase activity--along with cofactor 4B |
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translation pre-initiation of tRNA in eukaryotes
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eIF2-GTP-Met-tRNAi bind to 40S with assistance from eiF-1,3,5.
this forms the 43S preinitiation complex |
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translation initiation in eukaryotes
mechanism? |
take the 43S preinitiation complex and the activated mRNA complex together.
this makes the 48S initiation complex the mechanism of interaction is between 4G, 3, and the polyA tail 48S complex scans mRNA for AUG. when AUG is found, there is a "click" and eiF-1 dissociates causing GTP hydrolysis. this causes the release of all factors from the complex. eiF-2 has the GDP then 60S subunit joins. |
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why is the 3'UTR so important for translation?
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because of the polyA tail and its importance in combining the two pre-initiation complexes in the right spot. this brings the 3'UTR right at the beginning where its bound translation factors can work.
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why is eiF-1 so important?
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prevents the premature hydrolysis of GTP in the initiation complex
also releases mRNA from 40S subunit in termination |
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how is elongation and termination different in eukaryotes vs prokaryotes?
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euk has eF1 (like Tu and Ts of pro) and eF2 (like G of pro)
Erf1 (binds to all stop codons unlike Rf1 and Rf2 in pro) Erf3 (releases polypeptide in GTP dependent manner) in euk: the ribosomes are not dissociated by factors, rather the polypeptide is dissocated from the ribosome. |
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why is eiF-3 important?
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causes dissociation of 60 and 40S subunits in termination
also helps bind the tRNA complex to the mRNA complex in the right spot during initiation |
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reinitiation in eukaryotes
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takes place in the few polycistronic mRNA's
occurs after eiF-3 causes dissociation but before eiF-1 breaks apart the mRNA from the 40S. |
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how do viruses affect translation in eukaryotes while still allowing translation of their own proteins?
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first of all they inhibit eif-4G to stop initiation.
but they work because they have: 1) IRES site which binds with eiF-3 and bypasses the 4G requirement or 2) they have polyA tail at the 5' end which bypasses the need for 4G or 3 altogether! |
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regulatory role of eiF-2 phosphorylation
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three subunits:
alpha - gets phosphorylated beta - holds together gamma - interacts with tRNA, mRNA if phosphrylated, cannot initiate translation and all translation stops. |
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if only 30% of eiF-2 is phosphorylated, then why is 100% of translation inhibited?
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Because eIF-2B is needed to get rid of GDP and make GTP. and if the eIF-2 is phosphorylated, it will bind eif-2B and NOT LET GO.
there is a very small amount of eif-2B and once they are all bound up, 100% of translation stops |
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4 ways to phosphorylate eif-2
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1) hemoglobin pathway: excess heme activate kinase to phosphrylate eif-2
2)GAAC pathway: if there is not enough aa's, then there will be many empty tRNA molecules. the empty tRNA molecules active GCN2 which is kinase that phosphorylates eIF-2 until enough aa's are available to inactivate GCN2 (GCN4 still translates at a higher rate to produce more amino acids) 3) dsRNA activates PKR which phosphrylates eif-2 4) PERK (activated during unfolded protein response) can phosphrylate eif-2 |
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cells defense against dsRNA (3)
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1) dsRNA activates PKR which phosphrylates eif-2 to stop translation
2) activates oligosynthetase which activates a nonspecific RNAase to kill all RNAs 3) activates RNAi |
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how does dicer work?
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PAZ domain, 1 and 2 RNAase domains.
functions as a molecule ruler cuts down dsRNA into two short ds fragments with overhanging ends. dicer then carries the short ds fragments to RISC |
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what does RISC do?
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RISC gets short dsRNA from dicer and unwinds into guide/passenger strand.
the RNA with the less stable 3'OH overhang becomes the passenger. RISC+guide strand find complementary ssRNA and Ago2 subunit cleaves or blocks translation by binding to 3'UTR |
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what proteins are synthesized in the ER?
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secreted and membrane bound proteins
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why are stability requirements for proteins in ER so much higher then free ribosomal proteins
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because these proteins can leave the cell, so need to be more careful.
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how can secreted proteins be more stable
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disulfide bonds (reducing env)
glycosylated |
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how does cell know what protein needs to be secreted?
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signal sequence
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what is a signal sequence
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not a conserved sequence, rather a moderate hydrophobic region in the N terminus that signals the SRP to take the ribosome to attach to the ER.
it gets cleaved by signal peptidase once mature protein emerges in the lumen of RER. |
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SRP and how it stops translation
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signal recognition peptide
recognizes the signal sequence and carries the unfinished protein to the ER for release into the lumen consists of 7S RNA and p54 protein which has gtpase activity. p54 binds to the hydrophobic region and another subunit of SRP inhibits eF2 (translocation) and stops translation |
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how does SRP interact with ER?
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SRP interacts with the alpha receptor and hydrolyzes gtp.
this brings Sec61 translocon close to it and interacting with the ribosome so that it sits on top of it. lastly, the now hydrolyzed srp loses all interaction affinity with the ribosome and the srp receptor on the er |
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bip and grp94
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chaperones in the RER that are analogous to hsp70 and 90 respectively
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modification enzymes in RER
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chaperones (no hsp60 but yes hsp70-bip)
disulfide isomerase - to give disulfide bonds N-glycosylation |
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how does protein become transmembrane?
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during translation, a large hydrophobic sequence is translated and gets inserted into the membrane...no known mechanism...
can loop more then once. |
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type 1 vs type 2 transmembrane proteins
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type 1: N terminus is in the lumen
type 2: C terminus is in the lumen |
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N-glycosylation vs O-glycosylation
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N - done in the ER and on asn residue
dolichol phosphate attaches: 3 glu, 9 mannose, 3 n-acetylglucosamine to residue as it is translated O - done in golgi and on thr or ser residues |
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calnexin and calreticulum
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binds to sugar molecules on proteins in ER lumen
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what happens if protein folds incorrectly in ER?
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sugar molecules are cleaved off 1 at a time (3 glu and 1 mannose) and it is sent back out to the cytosol (ERAD)
after 4 times, it is sent to degradation (ubiquitin) |
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unfolded protein response
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1) ATF 6 sensor binds with BiP. when Bip levels go down (meaning lots of unfolded proteins), ATF 6 gets sent to golgi and TF region is freed to go to nucleus and activate BiP transcription.
2) IRE1 has sensor region that binds Bip. when bip decreases, IRE1 can dimerize and cross phosphorylate which activates the RNAase regions that splices mRNA for HAC1 (unusual since splicing occurs in nucleues only) the two exons are ligated by tRNA ligase. and HAC1 is now activated and is a TF for expression of Bip and grp94. also potentiates the erad pathway lastly, PERK is activated to stop translation |
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cop II
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proteins that coat the vesicles that transport between ER and golgi
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how do you know cells belong in ER
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they have KDEL sequence. if they get transported to golgi, then they get sent back to ER
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cop I
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proteins that coat vesicles that send between golgi and ER
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tag that sends proteins to lysosomes
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mannose-6-phosphate
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MSF
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mitochondrial import stijmulating factor.
recognizes the mitochnodrial import sequence: amphiphilic alpha helix with positive charges on one side and hydrophobic residues on the other side. brings the protein over to the mitochondria |
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what is the role of SRB?
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chromatin remodeler
co-activator helps with splicing |
