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117 Cards in this Set

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DNA Replication
-Bidirectional-occurs in both directions- but always 5'-3' because the strands are antiparallel, semi-conservative (one parent strand=template), and polarity based
Replication Origin
Between A and T because fewer hydrogen bonds
DNA polymerase
-Enzyme that allows DNA synthesis to proceed- acts as catalyst
-Synthesize a growing strand of DNA by adding bases that are complementary to a template strand
-Only works in 5'-3' direction
-Cannot work if the DNA that serves as the template is completely single stranded or completely doubled stranded
-Works only when 3' hydroxyl group and a single-stranded template are available
-Needs RNA
-Sometimes causes errors- GT, CA
-Self-corrects errors-proofreading (exonucleate activity) when it removes mispaired nucleotides in the 3'-5' direction
RNA Primer
-Needed for DNA polymerase to work
-Made by DNA primase
Replication fork
Y-shaped region where the parent DNA double helix is split into two single strands which are then copied
DNA Replication
-Bidirectional-occurs in both directions- but always 5'-3' because the strands are antiparallel, semi-conservative (one parent strand=template), and polarity based
Replication Origin
Between A and T because fewer hydrogen bonds
DNA polymerase
-Enzyme that allows DNA synthesis to proceed- acts as catalyst
-Synthesize a growing strand of DNA by adding bases that are complementary to a template strand
-Only works in 5'-3' direction
-Cannot work if the DNA that serves as the template is completely single stranded or completely doubled stranded
-Works only when 3' hydroxyl group and a single-stranded template are available
-Needs RNA
-Sometimes causes errors- GT, CA
-Self-corrects errors-proofreading (exonucleate activity) when it removes mispaired nucleotides in the 3'-5' direction
RNA Primer
-Needed for DNA polymerase to work
-Made by DNA primase
Replication fork
Y-shaped region where the parent DNA double helix is split into two single strands which are then copied
Helicase
Enzyme that catalyzes the breaking of hydrogen bonds between nucleotides in DNA causing the two strands of DNA to separate
Primase
Synthesizes a short strech of riobnucleic acid that acts as a primer for DNA polymerase
-type of RNA polymerase (an enzyme that catalyzes the polymerization of ribonucleotides to RNA)
-does not require a primer
Synthesis of leading strand
-once the primer is in place, DNA polymerase III begins adding deoxyribonucleotides to the 3' end of the new strand in a sequence that is complementary to the template strand
Sliding clamp
-doughnut shaped structure that holds the DNA polymerase in place as it moves along the DNA
-Made up of two subunits that come together
Synthesis of the lagging strand
-Synthesized in opposite direction of the replication fork
-Starts when primase synthesizes a short strech of RNA that acts as a primer
-DNA polymerase III then adds bases to the 3' end of the lagging strand-> enzyme moves away from the replication fork, helicase continues to open the replication fork and expose new single stranded DNA on the lagging strand
-DNA polymerase I removes RNA primer at the start of each fragment and fills in the appropriate deoxyribonucleotides
-DNA ligase catalyzes the formation of a phosphodiester bond b/w the bases synthesized by DNA polymerase-closes gap in sugar-phosphat backbond
Okazaki fragments
short DNA fragments involved in synthesis of the lagging strand
Single-strand DNA-binding protein
Proteins that attach to the separated strands and prevent them from snapping back into a double helix
-Allows strand to be straight- helps DNA polymerase
-Prevents premature rebinding of an adjacent strand
Replication Machine
Many proteins come together for DNA replication to occus: DNA helicase, single-strand DNA-binding protein, sliding clamp
Telomere
The region at the end of a linear chromosome
-Do not contain genes- They consist of short streches of bases that are repeated over and over
The end replication problem
Lagging strand- after primer removed-no DNA synthesis occurs- no free 3' end for DNA polymerase- results in unreplicated end and shortened chromosome
-Not a problem in bacteria bc cells have single, circular chromosome
Solution: Replication by telomerase:Catalyzes the synthesis of DNA from an RNA template. The enzyme carries an RNA molecule with it that acts as a built-in template. Th sequence of this RNA is complementary to the repeated sequence in telomere DNA- Can add repeats onto the end of a chromosome
-Binds to overhanging section of single-stranded DNA-extends sequence-primase,DNA polymerase, and ligase then synthesize the lagging strand in the 5'-3' direction restoring the original length of the chromosome
Methylation-Directed Mismatch Repair
-Repair proceeds only when methyl group is present
-Repair proteins always removed the mismatched base on the unmethylated strand
-Prokaryotes
Exonuclease
DNA polymerase enzyme removes nucleotides from DNA
-Removes only from 3' end of DNA and only if they are not H-bonded to a base on the complementary strand
-Thus- DNA polymerase can proofread-reduces a DNA polymerase's error rate to one per 10 billion bases
Eukaryotic Mismatch Repair
Enzyme that breaks covalent bonds creates a nick in the newly synthesized strand
-DNA mismatch repair proteins bind to mismatch
-Removes newly synthesized DNA strand
-Repair of gap by DNA polymerase and ligase
Depurination
Purine-base section (Guanine or Adenine) cleaved and produces depurinated sugar-
-Harmful-damages DNA
Deamination
Change from cytosine to uracil
-chemical modifactions of nucleotides, if left unrepaired, produce mutations
Excision Repair
-UV induced thymine dimers cause DNA to kink
Basics of DNA repair (Eukaryotes)
-Damage to top strand->excision of damaged region, DNA polymerase makes new top strand using bottom strand as a template-DNA ligase seals nick
Triplet Code
Three-base code that codes for amino acids-64 possibilities-so redundant-more than one triplet of bases might specify the same amino acid
Codon
Group of 3 bases that code for a particular amino acid
-Start codon: signals the start of protein synthesis on a certain point on the mRNA molecule (AUG-codes for methionine)
Stop codon: Signal that the protein is complete (UAA, UAG,UGA)
The Central Dogma
-Information flows from DNA to RNA to proteins (transcription, translation)
-DNA sequences define the genotype; proteins create the phenotype
-Changes in the genotype may lead to changes in the phenotype (transcription,translation to proteins)
RNA polymerase
Large, multi-protein complex, does the unwinding of DNA-binds to DNA
-Performs template-directed synthesis in the 5'-3' direction
-Does not require a primer to begin transcription
-Matches base in a ribonucleotide triphosphate with the complementary base in a gene
-Catalyzes the formation of a phosphodiester bond b/w the 3' end of the growing mRNA chain and the new ribonucleotide
-Newly syntheized RNA transcript leaves polymerase
-Active site: Where phosphodiester bonds form, contains Mg2+ which plays a role in stabilizing the transition state as the polymerization reaction takes place
Sigma
Detachable protein subunit that binds to RNA polymerase before transcription can begin
-Regulatory protein-responsible for guiding RNA polymerase to specific locations where transcription should begin
Holoenzyme
-consists of a core enzyme, which contains the active site for catalysis and other required proteins
-RNA polymerase +sigma
Promoters
Sections of DNA where transcription begins- where holoenzyme binds
mRNA
Codes for proteins
rRNA
Forms part of the structure of the ribosome and participates in protein synthesis
tRNA
Used in protein synthesis as adaptors b/w mRNA and amino acids
-Specific size- about 80 nucleotides
-Clover leaf shape-loops, neighboring single strands fro m H-bonds
-Tertiary structure important b/c it results in a precise separation b/w the anticodon and the attached amino acid
Small RNA
-Used in pre-mRNA splicing, transport of proteins to the ER, and other cellular processes
Initiation Phase of Transcription (Bacteria)
-Sigma binds to promoter (-35 and -10 boxes)
-DNA helix opens up and creates two strands of single-stranded DNA
-Template strand threaded through a channel that leads to the active site inside RNA polymerase
-Ribonucleoside triphosphates (NTPs) pairs w/ a complementary base on the template strand of DNA-begins RNA polymerization (NTP has potential energy due to phosphate groups)
Elongation Phase of Transcription (Bacteria)
RNA polymerase moves along the DNA template in the 3'-5' direction of the template strand, synthesizing RNA in the 5'-3' direction
-Enzyme's "zipper", a group of projecting amino acids, helps open the double helix at the upstream end
-Enzyme active site catalyzes the addition of nucleotides to the 3' end of the growing RNA molecule
Termination Phase of Transcription (Bacteria)
-Transcription stops when RNA polymerase reaches a strech of DNA sequence that functions as a transcriptoon termination signal
-As soon as it is synthesized, the RNA sequence folds back on itself and forms a short double helix held together by complementary base pairing-hairpin->disrupts interaction b/w RNA polymerase and the RNA transcript, resulting in the physical separation of the enzyme and its product
Basal Transcription Factors
Proteins that initiate eukaryotic transcription by matching the enzyme with the appropriate promoter region in DNA
-analogous to sigma proteins in bacteria
Prokaryotic vs. Eukaryotic Transcription
Bacteria: Single sigma protein binds to a promoter
-1 mRNA can make several proteins from multiple genes
-no processing (cap/tail)
Eukaryotes: Many basal transcription factors required to initiate transcription
-3 distinct types of RNA polymerase
-RNA pol II produces mRNA
-RNA pol I makes the large RNA molecules that are found in ribosomes
-RNA pol III manufactures small RNAs and tRNA
-more diverse promoters-TATA box
-1 mRNA can make 1 protein from 1 gene
-Cap/tail
DNA polymerase vs. RNA polymerase
DNA polymerase: Needs RNA primer
RNA polymerase: Can start from nothing, not as good at proofreading
Exons
Regions of eukaryotic genes that are part of the final mRNA
Introns
Untranslated sections of genes not represented in the final mRNA product
-tend to be longer than exons
Splicing
Introns removed from growing mRNA strand
-pieces of the primary transcript are removed and the remaining segments are joined together
snRNPs
Complex of proteins and small RNAs that catalyze splicing
-Bind to specific sequences on the primary RNA transcript that define the exon-intron boundaries
Primary RNA transcript
Contains both exon and intron regions
-Evolutionary advantage-longer the gene, more opportunites for crossing over and genetic diversity
Spliceosome
Multipart complex of inital snRNPs as well as new snRNPs
Splicing
After formation of spliceosome, the intron forms a loop with an adenine ribonucleotide at its base
-The loop breaks when the 2' hydroxyl group on the adenine attacks a phosphodiester bond at the 5' end of the intron
-Once the initial cut in the RNA is made, the free 5' end of the intron becomes attached to the adenine nucleotide and forms a lariat of RNA
-Then the free 3' end of the first exon reacts w/ the 5' end of the second exon-breaks the 3' end of the intron and covalently joins the two exons into a contiguous coding sequence
5' Cap
Structure added to 5' end of mRNA as soon as the 5' end of a eukaryotic mRNA emerges from RNA polymerase
-consists of the molecule 7-methylguanylate and 3 phosphate groups-methylated guanosine
-Serves as recognition signal for the translation machinery
-protect mRNAs from degradation by ribonucleases and that they enhance the efficiency of translation-also can produce more proteins
Poly (A)tail
Sequence at the end of the 3' end of the mRNA
-Formed when enzyme cleaves the 3' end and other enzymes add a long tract of 100-250 adenine nucleotides- adenosine sequence-polyadenylation, enzyme mediated process
-Extends the life span of an mRNA by protecting the message from degradation by ribonucleases in the cytoplasm
-protect mRNAs from degradation by ribonucleases and that they enhance the efficiency of translation-also can produce more proteins
-Functions as zip code in cell
-Tail varies in length, influences stability
Alternative Splicing
Used to produce tissue specific messages
-does not apply to all genes
-From one DNA sequence- can make many different types of mRNA->variation, 38,000 possible in fruit flies
-can make many proteins from splicing patterns of one gene
-isoforms-came from same gene
-Controlled by regulatory proteins that bind to mRNA in the nucleus and interact with spliceosomes
Export of mature mRNA
-Poly-A-binding protein binds to the tail to make sure it is ready for export
-Only "export ready" RNA will come out-through nuclear pore complex w/ help of RNA export factor
-mRNA has certain life span- if not stable enough, will be degraded by cell quickly- eventually all will be degraded
Ribosomes
-Site of protein synthesis
-Strong, positive correlation b/w ribosomes in a cell and the rate at which that cell synthesizes proteins
-Attach to mRNAs and begin synthesizing proteins even before transcription is complete
-Where mRNA message is decoded
-located on edge of ER (rough ER) or in cytoplasm
Bacteria: no nuclear envelope so transcription and translation can occur concurrently
Eukaryotes: mRNAs are spliced in the nucleus and then exported to the cytoplasm. Once RNA messages are outside the nucleus, ribosomes attach to them and begin translation- thus separated
Aminoacyl tRNA
A tRNA molecule that becomes covalently linked to an amino acid
-requires input of energy from ATP
Aminoacyl tRNA synthetases
Enzymes that are responsible for catalyzing the addition of amino acids to tRNAs
Anticodon
Set of 3 nucleotides that forms base pairs with the mRNA codon
-Each tRNA has a distinct anticodon and attached amino acid- but same L-shaped structure
-Always written in 3'-5' direction
-Complementary to codon- end of cloverleaf
-binding site for mRNA codon
A site
tRNA that carries the amino acid is bound here
-Aminoacyl tRNA diffuses into A site and its anticodon binds to a codon in mRNA
A= acceptor or aminoacyl
P site
tRNA that holds the growing polypeptide is bound here
-A peptide bond forms b/w the amino acid held by the aminoacyl tRNA and the exisiting polypeptide, which is held by the tRNA in the P site
P= peptide bond formation
E site
tRNA that no longer has an amino acid attached and is about to leave the ribosome
-The ribosome moves ahead and all 3 tRNAs move one position down the line. The tRNA in the E site exits; the tRNA in the P site moves to the E site; and the tRNA in the A site switches to the P site
E=exit
Initiation (Translation)
-mRNA binds to the small subunit w/ translation initiation factors bound to the 5' cap on the mRNA of the ribosome
-Initator tRNA bound to methilyne amino acid (AUG start codon)
-Initation factors dissociate-> large subunit binds
-In prokaryotes:no RNA cap to recruit small ribosomal subunits-instead has ribosomal binding sites
Elongation (Translation)
-At the start.. E and A sites in the ribosome are empty of tRNAs
Aminoacyl-tRNA binds to the codon in the A site via complementary base pairing b/w anticodon and codon
-tRNA occupies both P and A site-peptide bond forms catalyzed by RNA (ribozyme)-requires energy
-Shifts tRNA from A site to P, and P site to E-> caused by conformational change (5'-3' direction)-Translocation
-New peptide bond forms
-Released at Esite -uncharged
Termination
-3 stop codons in genetic code- no anticodon can bind to these sequences
-When stop codon exposed, a protein called a release factor fills the A site- active site catalyzes the hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain
-Frees polypeptide and it is released from the ribosome. Cleaved peptide bond forms C terminus
-Then ribosome separates from mRNA and the 2 ribosomal subunits dissociate
Polyribosomes
Many ribosomes read 1 mRNA molecule
-Ribosomes move down mRNA chain- more efficient and can translate more proteins
-Used by both prokaryotes and eukaryotes
Post-translational Modifications
Molecular chaperones-proteins that speed up folding process
-folding, addition of carbohydrate or lipid groups, phosphorylation
-activates protein
Antibiotics
Prohibit protein or RNA synthesis (transcription/ translation)
i.e. tetracyclin blocks binding of aminocyl tRNA to A site of ribosome
Cell differentiation
Cell that gives rise to many cells- each have their own function and protein expression pattern
-Differentiated cells capable of cloning b/c all cells share the same DNA
Transcriptional Control
The cell could avoid making the mRNAs for a particular enzyme-thus ribosomes cannot make the gene product
i.e. Various regulatory proteins affect the ability of RNA polymerase to bind to a promoter and initiate transcription
-Very efficient-saves the most energy bc it stops the process at the earliest point-very slow however
Translational Control
-Most common mechanism- but most common and lengthy step-able to change quickly which proteins are produced
-Mechanisms that alter the length of time an mRNA strand survives before it is degraded by ribonucleases, that affect translation inititation, or that affect elongation factors and other proteins during the translation process
Post-translation Control
-Some proteins are manufactured in an inactive form and have to be activated by chemical modification, such as the addition of a phosphate group
-Most rapid response- but energetically expensive
Inducer
Molecule that stimulates the expression of a specific gene
Gene regulatory proteins
Activators (DNA binding proteins) or repressors
-not an all or none event- can be intermediate expression
Operator
The binding site for the repressor
-found in promoter sequence
Repressor
Protein that can inhibit or decrease expression by binding to DNA and preventing transcription (Prevents binding of a polymerase to the promoter)
Basal level of transcription
Operater can stimulate such expression (low level)
Constitutively
Genes- such as those that code for enzymes required for glycolysis- are transcribed all the time
Activator
Found upstream at activator binding site-> Spontaneous isomerization leading to activated level of transcription
-Acts as a recruiter- allows/facilitates binding of RNA polymerase
Polycistronic Gene
Bacterial genes- Many genes-1 RNA molecule- Many enzymes (a single mRNA may contain several start/stop codons and ribosome binding sites and thus code for several distinct proteins)
-i.e.-Operon all controlled by a single promoter
-eukaryoes: I gene, 1 promoter
-prokaryotes: Many genes, 1 promoter
-i.e if excess tryptophan- binds to repressor making it active- example of allosteric regulation
lac Operon
3 genes-
lac Z-Codes for enzyme B-galactosidase- only signaled when lactose is high and glucose is low- needs permease from lacY
lac Y-Encodes for galactoside permease- membrane protein responsible for transporting lactose into the cell
lac I: Encodes for repressor protein that binds to DNA and prevents transcription of lacZ and lacY- expressed constitutively
CAP
-Regulates transcription- activator- CAP binds to sequence- protein interacts w/ RNA polymerase in a way that allows transcription to begin much more frequently-exerts positive control on the lac operon
-allosterically regulated-CAP changes shape when cAMP binds to it and causes it to bind to DNA
Positive Control of the lac Operon
-When something must be added for transcription to occur
CAP only activates when glucose is low- need lactose to inhibit the repressor
-Typically- glucose inhibits the activity of the enzyme
-Depends on transcription activator proteins-such as CAP
Glucose +Lactose
Operon off b/c CAP not bound- glucose inhibits enzyme adenylyl cyclase which produces cAMP- necessary for CAP to bind
Glucose but no Lactose
Operon off b/c lac repressor bound and b/c CAP not bound
No glucose or lactose
Operon off b/c lac repressor bound (lactose deactivates repressor)
Lactose but not glucose
Operon on-> Repressor cannot bind bc of presence of lactose so RNA polymerase can bind. cAMP present so binds to DNA
Negative Control
When something must be taken away for transcription to occur-i.e lac I gene produces an inhibitor that exerts negative control over lacZ and lacY genes
-Based on repressor proteins-such as lacI product
cAMP vs. Glucose
Glucose conc high-cAMP conc low
-driven by the enzyme adenylyl cyclase which produces cAMP from ATP
-Adenylyl cylase's activity is inhibited by extracellular glucose
Structural Motifs
Proteins recognize DNA via specfic secondary stuctures (domains)
-alpha helix best to react with
-Also: Beta-sheet binding, leucine zipper, zinc finger
Recognition Sequence
The section of the helix-turn-helix domain that binds inside the major groove (half-site)
-Different h-bond acceptors and donors- strength of binding
-Chemical greups exposed from edges of base pairs-> Creates different types of bonds, covalent, ionic-> very specific
*Can have DNA binding- but not activated*-There is a DNA binding region and activator region
Dimerization
-2 Seperate proteins connected through phosphorlyation
Binds to 2 different regions-less room for errors- more accurate -> Specificity
-More stable due to stronger interactions
Domains
Regions on proteins that have distinctive 3D structure and function
Anatomy of DNA
-Must have a promoter->Where RNA polymerase and basal transcription factors are recruited
-Close by are promoter-proximal elements(Where regulartory TFs bind)
3 Main Factors of regulation in eukaryotes
1. Basal Transcription Factors
2. Regulatory TFs
3. Chromatin
Chromatin
DNA wrapped around proteins to creat protein-DNA complex
Chromatin-remodeling
Before transcription can begin in eukaryotes, DNA near the promoter must be released from tight interactions with proteins so that RNA polymerase can make contact with the promoter
RNA processing
The steps required to produce a mature processed mRNA from a primary RNA transcript
Histones
DNA-associated proteins
activated chromatin-> acetylated histones (HATS)
deactivated chromatin-histone deaceylases (HDACS)
-Postively charged b/c of many lysine and/or arginine residues
-Binds w/ DNA bc of its negative charge due to phosphate groups
Nucleosome
"beads"
-DNA wrapped almost twice around a core of 8 histone proteins
-B/w each pair of of nucleosomes there is a linker strech of DNA associated with a histone called H1
Chromatin-remodeling complexes
Group of proteins which reshape chromatin through a series of reactions that are dependent on ATP
Acetylation
Chromatin-remodeling protein that adds acetyl groups to histones
-Results in active gene expression-> Recruits proteins
Methylation
Chromatin-remodeling protein that adds methyl groups to histones
-Results in gene silencing
Histone Acetyl Transferases (HATS)
Acetylate the positively charged lysine residues in histones
-When a HAT adds an acetyl group to selected histones, the # of positive charges on the histones is reduced- Result=less electrostatic attraction b/w histones and the neg-charged DNA. Thus the association b/w nucleosomes and DNA is loosened and the chromatin decondenses
-On switch for transcription
Histone Deacetylases (HDACs)
Group of enzymes which remove acetyl groups added by HATs. Histone deacetylase activity reverses the effects of acetylation, returning chromatin to its default condensed state
-Off switch for transcription
TATA-binding protein
Protein that binds to all eukaryotic promoters
-Recruits RNA polymerase and Basal TFs
Regulatory sequences
Sections of DNA that are involved in controlling the activity of genes, similar to the CAP site and operators
Promoter-proximal Elements
Regulatory sequences located close to the promoter and bind regulatory proteins
-Have sequences unique to specific genes
Enhancers
Regulatory elements that are far from the promoter
-Can be more than 100,000 bases away from the promoter. They can be located in introns or in untranscribed 5' or 3' sequences flanking the gene
-Like promoter-proximal elements, many types of enhancers exist. Different genes are associated with different enhancers
-Enhancers can work even if their normal 5'-3' orientation is flipped
-Enhancers can work even if they are moved to a new location in the vicinity of the gene, on the same chromosome
-Where ACTIVATOR binds
Silencers
Where REPRESSOR binds
-When silencers are active, they shut down transcription
-Opposite in function to enhancers
How do enhancers work?
In multicellular species, different types of cells express different genes bc they contain different regulatory proteins
-Regulatory proteins bind to specific enhancers and silencers and promoter-proximal elements- trigger production of muscle-specific proteins
Regulatory Transcription Factors
Proteins that bind to enhancers and promoter-proximal elements
-Responsible for the expression of particular genes in particular cell types and at particular stages of development
-When they bind to DNA they recruit chromatin-remodeling complexes and HATS
Co-activators
Involved in starting transcription
-Do not bind to DNA
-Link proteins involved in initiating transcription-regulatory transcription factors and basal transcription factors
Basal Transcription Complex
Multi-protein machine composed of basal transcription factors that have assembled at the promoter in response to interactions with regulatory transcription factors and co-activators
-Construction begins when TBP binds to the TATA box, then as many as 60 proteins assemble around DNA-bound TBP
-Able to position RNA polymerase II in a way that initiates transcription
Post-translational control
A group of regulatory transcription factors that is found in mammals-called signal transducers and activators of transcription (STATs)- participate in several signal transduction pathways
Epigenetic Control
Another way to regulate gene expression above gene sequences-i.e. chromatin structure, acetylation of histones, alternative splicing
-no need to do everything from scratch
Dimer
Formed when an activated receptor adds a phosphate group to a STAT polypeptide chain
-Activated STAT dimer then moves to the nucleus, binds to an enhancer, and activates transcription