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113 Cards in this Set
- Front
- Back
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genome
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all genes in organisms DNA
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proteome
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all encoded proteins.
The proteome is not all expressed at once. The proteins are expressed at specific time and place to allow organism to devolop, reproduce, and die |
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describe protein polypeptide bond
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carboxyl group of alpha carbon of one amino acid in covlently bonded to amino group attached to a carbon of another aa.
amino group always on left and carboxy terminus is always on the right |
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what accounts for almost all the DNA in the bacterial chromosome?
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genes and regulatory sequences
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are bacterial genes haploid or diploid?
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Haploid so if something goes wrong, they just die
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how is mRNA transcribed? What does RNA polymerase act on?
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The RNA polymerase acts on the template strand to transcribe mRNA so the mRNA is the same as the coding strand.
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what are structural RNA
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genes encode RNA used during translation that are NOT translated themselves. Ex: ribosomes, rRNA, tRNA
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componenets of RNA polymerase
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4 genes alpha, beta, beta prime, and omega
5 polypeptides though 2 alpha, 1 beta, 1 beta prime, and 1 omega therefore, alpha pp needs to be made at a rate 2x that of the others bc need excess alpha pp to make RNA polymerase |
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What two fundamental processes are required of DNA and of RNA?
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DNA: Replication and transcription
RNA: Transcription and translation |
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transcription
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making RNA copy of a gene from DNA
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Translation
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makes protien from mRNA using tRNA and ribosomes containing rRNA
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what are 2 things viruses does that diverge from the central dogma of molecular biology?
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RNA replication, the RNA virus can make anothe copy of RNA
Reverse Transcription (HIV) the RNA uses reverse transcriptase to make DNA. So viral DNA invades the host cell and makes viral proteins |
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viral genome
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RNA then use RNA relicatoin and reverse transcription to make DNA
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What accounts for an organisms developmental and phenotypic characteristics?
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Gene expression
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What does protien synthesis involve?
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transcription and translation. A gene is transcribed to mRNA and mRNA is translated to polypeptide using tRNA and rRNA
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important features of the structure of RNA
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ribose has OH at 2' position
bases have uridine instead of thymidine RNA is single stranded RNA can be hydrolyzed by alkali RNA has complementary regions that can fold back on itself making a 3D double helical region (ribosomes, tRNA) |
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RNA hydrolysis
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use alkali so the 2' OH acts like a nucliophile. A OH takes the H off of the 2'OH making a phoshodiester bond between 2'and 3' and breaking the 3'->5' phosphodiester bond
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can tRNA and rRNA be hydrolyzed by alkali
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no bec they have proteins to protect them from hydrolysis
alkali hydrolysis degrades mRNA mainly |
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What are the requirements of DNA dependent RNA polymerase?
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a) Core enzyme
b) DNA strands, c) Mg & Zn ions, d) all 4 NTPs |
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What ions are needed for DNA dep. RNA polymerase?
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Zn ion for NTP binding and 2 Mg ions for catalysis
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In which direction is mRNA syntheisized?
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from 5' to 3'.
Catalysis is by 3'OH attacking 5'phospate of incoming NTP |
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The RNA polymerase core enzyme has 4 subunit polypeptides, alpha, beta, beta' and omega. What are their functions?
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Alpha – assembles enzyme and regulatory proteins to activate catalysis
Beta – catalysis; chain intitiation and elongation Beta’ – binding to DNA template Omega – chaperone; restores denatured enzyme |
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How does RNA polymerase extend a chain? What metal ion is essential for catalysis
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The chain extends by nucleophilic attack of the previously attached ribose 3' OH group on an incoming nucleotide triphosphate which therefore adds to the 3’end. Thus, the new chain grows from 5’ to 3’. Catalysis requires Mg+2 ions to coordinated to asparte residues on the enzyme
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describe transcription bubble
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RNA polymerase unwinds the leading strand to expose the template strand so it can synthesize mRNA from 5' to 3'. mRNA grows at the 3' end
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describe 5th subunit of RNA polymerase
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it is sigma. It is not encoded at the same time it is just transiently associeated with the other subunits. It sends the enzyme to the promoter
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What sends RNA polymerase to the promotor region?
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sigma subunit so RNA polymerase has to find the sigma factor before transcription can occur
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chaperone
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so Enzyme is held in correct conformation
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Does RNA polymerase have 3' to 5' exonuclease?
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NO only in DNA polymerase
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Where does RNA synthesis start and stop?
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starts at a promoter sequence and ends at a terminator sequence W/O these mRNA cannot be transcribed
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importance of RNA polymerase sigma factor
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allows RNA polymerase to bind to specific site on DNA. WO the sigma RNA polymerase binds everywhere
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promotor
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specific DNA sequences that determine where RNA polymerase will bind
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How are promotor sequences identified?
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Withe sigma subunit of RNA polymerase. The promotor has certain consesus sequence that the sigma recognizes and binds to.
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Why can promotor sequences be transcribed?
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bc the gene is downstream from the promotor
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When does sigma subunit fall away from DNA?
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when the RNA polymerase core enzyme binds and the DNA opens (beginning of transcription)
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What is a TATA box and why is it not transcribed?
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It is a conserved short DNA sequence required for sigma-70 or TATA-binding protein (TBP) subunit of RNA polymerase to bind and separate the strands. It is on the coding strand. RNA polymerase only transcribes from the template strand downstream of where sigma-70 or TBP first bound to the coding strand
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Where does sigma 70 bind?
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binds to -35 region TG box and -10 TATA box
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what effects the effciency of which RNA polymerase binds to a promotr
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determined by -35 and -10 sequences, the spacing between them and their distance from the transcripton start site
enhancers increase the ability of sigma binding to DNA |
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describe initiation and elongation
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sigma binds to tata and TG region. the core enzyme comes in (sigma + core enzyme make up holoenzyme) the DS DNA opens up and transcription starts. The sigma falls away. Enhancer causes RNA poly to bind tight to DNA so elongaton occurs quicdly. RNA polymerase add a base to the 3' end. mRNA synthesized from 5' to 3'
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termination of transcription
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At certain CA or AAA spequences on DNA the RNA polymerase binds "too" tightly causing a pause. The RNA either folds over on itself making it DS so if the RNA poly falls apart or Rho dependent protien binds to RNA at a consesus sequence and moves 5' to 3' to stop the complex
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Summary of steps in bacterial RNA synthesis
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transcription starts when sigma factor binds to promotor
RNA core polymerase binds to sigma and unwinds DNA transcription bubble forms and sigma falls away RNA bases are selected by hybridizing to the DNA template and extending 3' end Single RNA strand has same sequence as DNA coding strand (complementary to template) Two termination sequences: Hairpin (self complementary) or rho binding protein |
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How do the sigma (σ) and rho (ρ) proteins function?
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Sigma binds promotor sequences on DNA to initiate RNA synthesis
Rho binds terminator RNA sequences and this stops RNA synthesis |
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Indicate three ways in which the process of transcription is more complex in eukaryotes than in prokaryotes?
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a) Different polymerases for different types of RNAs
b) Many additional accessory protein factors c) Core enzyme has additional C terminal domain (CTD) that gets phosphorylated |
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Describe three characteristics of a prokaryotic mRNA transcript that differentiates it from a eukaryotic transcript?
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Prokaryotic mRNA transcripts have no introns, are not processed and are translated immediately because they are rapidly degraded
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Actinomycin D inhibits transcription in all organisms. Compare its mode of action with that of rifampicin and α amanitin?
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Actinomycin D inhibits all RNA transcription because it binds to certain (GpC) sequences in DNA, preventing the open transcription complex from forming. Rifampicin binds to the active site of bacterial but not eukaryotic RNA polymerase, whereas α amanitin binds strongly to RNA Pol II, inhibiting eukaryotic but not bacterial mRNA transcription
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RNA pol I
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transcribes ribosomal rRNA in the nucloplasm
the rRNA is not translated |
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RNA pol II
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transcribes pre mRNA in nucleoplasm.
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How does eukaryotic RNA poly II differ from prokaryotic RNA poly?
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the Pol II attaches more polypeptide cofactors than bacterial RNA poly and recognizes promotors that are much more vaired in sequence than bacterial promotors
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RNA pol II
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transcribes tRNA and small RNAs neither are translated
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RNA pol IV
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Transcribes mitochondiral RNA (related to bacteriophage enzyme)
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Antinomycin D
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inhibits both bacterial and eukaryotic RNA elongation bc it is similiar to nucleotide base and it interferes with GpC sequences and prevents replication bubble from forming so DNA doesnt seperate and RNA pol cant work
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What inhibits RNA pol I?
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Nothing
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RNA pol II inhibitor
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low does of alpha amanitin which binds to euk RNA pol II and stops mRNA production
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What inhibits Bacterial RNA polymerase?
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antibiotic rifampicin
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describe how pol II works
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cell is signaled to start initiation of transcription bc TF binds to signal and then recruits more TF to bind to the exposed promotor when attracts pol II enzyme. The complex opens, the CTD gets phosphorylated. TF fall off. Elongation occurs until termination. At termination the RNA pol II is release and CTD is dephosphoylated
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Function of CTD on RNA polII
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involve in transcription activation and elongation and mRNA processing
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Name the 5 types of RNA in a eukaryotic cell, ordered from greatest to least amounts present. Which of the is absent from prokaryotes? Why?
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rRNA > tRNA > mRNA > snRNA > scRNA
lots of rRNA bc protein syntheis happeing all the time not much mRNA bc it is so stable it can be used over and over again snRNA is not in prokaryots because there processing of bacterial mRNA |
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what activates aa and incorporates them into pp by reading the genetic code on mRNA
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tRNA
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what RNA has many proteins incormporated with it. platforms for translation interaction of tRNA and mRNA and catalyzes peptide bond formatoin
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rRNA
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snRNA
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small nuclear RNA
in ribonucleoprotein that excises spliceosomes from primary mRNA transcripts (removes introns) |
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scRNA
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small cytoplasmic RNA; 2 kinds
1.moves newly synthesisized pp into ER lumen 2.acts as silencer to prevent sigma or TF from binding |
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2 functions of processing the eukaryotic mRNA
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1. splice out introns and put exons back together
2. stabilizes completed mRNA to go to the cytoplasm by adding poly guanine cap and poly A tail |
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5 types of eukaryotic RNA and their main function
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mRNA-messenger for pp
rRNA-binds proteins to form ribosome tRNA-transfers aa snRNA-most form splicesomes scRNA-export to ER, protein synthesis inhibitors |
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function of poly A tail
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stabilizes mRNA to allow it to go into the cytoplasm. it protects the mRNA from nuclease hydrolysis
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describe cap formation
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remember that the beginning of the 5' end of mRNA has 3P so use phosphohydrolase to revove 1 phosphate from 5' terminus
use guanylytransferase to remove another 5' phosphat as pyrophosphate and add FMP to 5' terminal diphosphate nucleotide guanine methyltransferase adds methyl to guanine Omethyltransferase adds methyl to first or second nucleotide of pre mRNA |
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4 enzymes in cap synthesizing complex
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1. phosphohydrolase
2. guanylyltransferase 3. guanine methyltransferase 4. o methyltransferase |
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What enzymes are required to process mRNA?
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a) Phosphohydrolase, guanosyl transferase and methylase (cap)
b) Polyadenylate polymerase complex containing endonuclease and polyA polymerase (tail) c) Small nuclear RNA particles with consensus binding sequences (spliceosomes) |
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how is poly A tail added to mRNA
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after an AAUAAA consensus is exposed downstram by 3' end. polyA polymerase enzyme complex binds to it and uses endonuclease to cleave downstream from consensus site. The polymerase complex adds many ATP molcules to make a poly A 3'tail
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Where are the consensus residues that identify an intron and are necessary for its removal from freshly synthesized RNA?
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The first two residues at the 5’ end, the last two at the 3’end and an adenosine residue about two-thirds of the way towards the 3’end.
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What enzymes are required to process freshly synthesized tRNA and rRNA?
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Specific endo and exo nucleases that trim the ends and may also be required to remove an intron
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how does mRNA splicing occur?
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small nuclear RNA (U) binds to certain consensus sequences found around introns at 5'end, 3'end, and a v. important adenosine 2/3 way to 3' end. the adenosine cuts off 5' end to form a noose and that activates the removal of the 5'end
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What is the RNA in spliceosomes called?
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Spliceosomes are composed of snRNA particles
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Why are these RNAs not found in bacterial cells?
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Bacterial cells have no introns
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What enzyme do snRNA attached to during splicing?
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Spliceosomes attach to RNA pol II
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What are the products of mRNA splicing?
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Two connected exons plus the excised intron in the form of an RNA lariat
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What are the functions of the cap and tail in eukaryotic mRNAs?
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The cap facilitates splicing The tail facilitates mRNA transport out of the nucleus Both facilitate the initiation of translation by attaching to the ribosomal small subunit
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Where does U1 and U2 bind?
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U1 binds to 5'end
U2 binds to the adenosine the binding of both cause intron removal |
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when do spliceosomes bind to mRNA?
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snRNAs attach to the posphorylated CTD after cap formation. splicesomes and cap bind during elongation.
When the poly A tail is added, the CTD is dephosphorylated and RNA poly II falls apart |
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3 roles of guanine cap
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1. splicing
2. go from initiation to elongation by phosphorylating the cap 3. makes and binds the cap |
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processing of tRNA
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tRNA folds in cloverleaf structure bc of internal complementary and has modified areas. enzymes recognize the modified areas and remove introns
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where are all preRNA transcripts processed?
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nucleus
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how does cap initiate s;icing?
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by binding the C terminal domain of Pol II where spliceosomes attach to a forming mRNA
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what causes diff pp from a single pre mRNA transcript?
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different splicing
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what allows the mRNA to exit the nuclear membrane and go into the cytoplasm
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the poly A tail
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type of RNA with a variable half life that is easily degraded and unstable
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most RNA is protein bond and stable except for mRNA which has a variable half life and is degraded
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One gene can encode two different polypeptides. How?
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1) Alternative splicing
2) Limited proteolytic processing |
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What is a ribosome
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Ribosome is an RNA protein particle on which protein are synthesized
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What is a proteasome
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Proteasome is a protein particle that digests ubiquitinylated proteins in the cytosol
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What is a spliceosome
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Spliceosome is an snRNA particle that removes introns as eukaryotic mRNA is being synthesized
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What is a exosome
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Exosome is a protein exonuclease particle that digests all types of RNA from 3' →5'
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In eukaryotic cells, where are ribosomes and proteins synthesized?
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Ribosomes are made in the nucleolus of the nucleus and proteins in the cytosol, often attached to the rough endoplasmic reticulum
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Why is the genetic code composed of trinucleotide sequences and not dinucleotide or tetranucleotide sequences?
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Dinucleotides have only 16 possible combinations, not enough to encode 20 different amino acids. Tetranucleotides have 256 possible combinations, far too many possibilities
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Why is codon degenerate:
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bc most aa are enceded by more than one triplet bc only 20 aa and with triplet codon have possibility of 64 codons
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With what was polyU incubated and how did that indicate its function?
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It was incubated with the alkali treated cytosolic fraction of either E coli, or liver cytosol that contained polyribosomes (microsomes; 20,000 g supernatant). The sediment was radioactive only if radioactive phenylalanine was used this indicated that UUU coded for phenylalanine
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How were specific sequences of polynucleotides shown to encode specific amino acids prior to elucidating the genetic code? Hint: Polynucleotide phosphorylase
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Polymers were made from single NDPs or pairs of NDPs using polynucleotide phosphorylase. Incubating ribosomes from the alkali-treated cytosolic fraction of E. coli with a high magnesium chloride concentration gave amino acid polymers whose composition depended on the nucleotides used and their proportion in the polymer
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AAA
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lysine
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CCC
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proline
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2 functions of AUG codon?
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1. starts protein synthesis
(formyl methionine) 2.makes methionine |
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stop codons
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UAA
UAG UGA |
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why is there way less tRNAs than codons?
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bc of the wobble position on the anticodon of tRNA
only codons that differ in either of the first two bases require different tRNAs |
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tRNA purpose in translation
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tRNA is an adaptor molecule that recognizes codons and inserts the aa into the pp in the right sequence
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ribosomal subunits of prokaryotes
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the 70S particle is made of 2 subunits. 30S and 50S are formed by ribosomal proteins associating with rRNA spliced from precursor in nucleolus then the subunits are exported into the cytosol
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where does synthesis, processing, and assembly of eukaryotic ribome subunits take place?
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in nucelolus and then they are exported to cytoplasm
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what happens to subunits in nucleolus?
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the subunits are assembled and remodeled using ATPase associated with various cellular activies that are imported with ribosomal proteins and sent to cytoplasm
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where mRNA interacts with the ribosome and tRNA to synthesize a polypeptide
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polysome
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How is amino acid activation for polypeptide synthesis accomplished?
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Activation is achieved by amino acid synthetase that bind ATP, a specific amino acid and a specific tRNA. Reaction produces pyrophosphate (PPi).
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Why are there two classes of amino acid tRNA synthetases?
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Enhance synthetase specificity for the correct amino acid
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Why is the genetic code not overlapping?
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There would be insufficient room for aligning the aa tRNAs next to each other to induce peptide bond formation if codons overlapped
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Why was the ‘wobble’ hypothesis necessary and what does it apply to?
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It is necessary because most amino acids are specified by more than one codon It applies to mRNA/tRNA codon/anticodon interactions on the ribosome during protein synthesis
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What are ribosomes and how are they categorized?
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Ribosomes are particles composed of structural RNA (no protein encoding function) and protein made in the normal way. The sizes of ribosomal particles or their component RNAs are measured in Svedbergs, a sedimentation coefficient measurement
the smaller the particle the slower it sediments and the smaller the S |
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What enzyme synthesizes eukaryotic ribosomes?
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The DNA genes encoding eukaryotic ribosomal RNA are transcribed by RNA polymerase I
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unusual bases of tRNA
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dihydrouridine (dehydrated uridine)
pseudouridine(isomer of uridine) ribothmidine |
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What are the 4 main features of a tRNA molecule, and their functions?
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a) The amino acid attachment arm – made of ACC at the 3’ end is recognized by amino acyl synthetase AND peptidyl synthetase
b) The DHU arm – sandwiches over the pseudouridine loop when changing from cloverleaf to L-form c) The pseudouridine arm d) The anticodon arm – interacts with mRNA extra arm adds variability but not always present |
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What is the significance of the tRNA L form?
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tRNA L form spreads out the tRNA across the ribosome, connecting the complementation to the mRNA with the peptidyl synthetase reaction
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