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58 Cards in this Set
- Front
- Back
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How do eukaryotes cope with their huge genome size to increase replication efficiency?
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-more than one chromosome --> multiple oriC's
-many oriC's per chromosome, all bidirectional |
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What is the function of the Origin Recognition Complex (ORC)?
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Origin Licensing:
ORC Binds oriC, recruits Cdc6 and Cdt1, MCM (minichromosome maintenance) loading proteins |
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What is the function of Cdc6 and Cdt1 at the origin of replication?
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Origin Licensing:
Recruit and load MCM (minichromosome maintenance) protein onto the origin. Origin is now licensed. |
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What helps to ensure that origin licensing occurs only once per origin per cell cycle?
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one ORC per origin
one set of Cdc6, Cdt1 per origin MCM, Cdc6 and Cdt1 are all destroyed after one use. |
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What is the role of the MCM complex at the origin of replication?
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Origin Licensing:
hexameric ring, latches onto the DNA |
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What is the function of cohesin at the origin of replication?
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-movable rings encircle DNA
-hold replicated daughter strands together until they are pulled apart during anaphase. loaded onto DNA with Cdc6/Cdt1/MCM. |
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What occurs in M phase of the cell cycle?
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mitosis: chromosomes condense, cell divides
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What occurs in G1 phase of the cell cycle?
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growth #1 phase: cell performs check prior to synthesis of DNA.
all clear --> ORC, Cdc6, Cdt1, MCM, cohesins loaded |
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What occurs in S phase of the cell cycle?
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Synthesis of DNA (replicate genome)
Cdc6, Cdt1, MCM all destroyed |
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What occurs in G2 phase of the cell cycle?
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Cell checks its work before initiation of mitosis
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What is the difference between prokaryotic and eukaryotic primers?
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pro: RNA only
euk: RNA primer + short DNA primer formed by error-prone DNA pol alpha |
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How are eukaryotic primers laid down?
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1. RNA primer laid down
2. primer extended by DNA pol alpha |
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What is the function of eukaryotic DNA pol alpha?
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error-prone DNA pol
part of primosome extends RNA primer no proofreading! |
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What is the function of eukaryotic DNA pol delta?
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synthesizes Okazaki fragments on lagging strand
can proofread major replicative polymerase. |
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What is the function of eukaryotic DNA pol epsilon?
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synthesizes the leading strand of DNA.
major replicative polymerase. can proofread. |
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How are eukaryotic primers removed?
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1. RNAse H1 (endonuclease-1) removes RNA primer
2. Flap Endonuclease (FEN) removes error-ridden DNA pol alpha primer only 5' end of the DNA synthesized using that removed primer remains; gets ligated to end of next okazaki. |
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What ions are required for RNA polymerase's catalytic activity?
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2 x Mg2+
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What is the regulatory advantage of having RNA and DNA?
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-regulate amount of RNA present via promoter strength --> regulate amt of protein
-allows gene amplification -can keep DNA "master copy" safely in nucleus |
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What is the function of RNA pol I?
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makes most rRNA
lives in nucleolus where ribosomes are made |
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What is the function of RNA pol II?
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lives in nucleoplasm
makes mRNA |
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What is the function of RNA Pol III?
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Lives in nucleus
makes tRNA + other small RNA molecules |
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What are the locations of the prokaryotic and eukaryotic TATA boxes?
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Pro: -7 to -10
Eu: -20 to -30 |
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Eukaryotic Transcription Initiation
What is the role of TFIID? |
TFIID: has TATA Binding Protein (TBP)
TBP introduces 45-degree bend in DNA, unwinds it |
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Eukaryotic Transcription Initiation
What is the role of TFIIB? |
Directs DNA's path through transcription complex.
Processivity factor: Stabilizes TFIID on DNA. |
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Eukaryotic Transcription Initiation
What is the role of TFIIA? |
Processivity factor (stabilizes TFIID on DNA)
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Eukaryotic Transcription Initiation
What is the role of TFIIE? |
Recognizes closed complex (no bubble yet)
Recruits TFIIH |
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Eukaryotic Transcription Initiation
What is the role of TFIIF? |
Melts DNA to form open complex
Helps keep open complex stable |
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Eukaryotic Transcription Initiation
What is the role of TFIIH? |
Helicase: stays at leading end of RNAP and unwinds DNA ahead of it.
introduces negative superhelicity into promoter DNA. |
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Eukaryotic Transcription Initiation
What is the role of the Enhancer sequence? |
-may be upstream, downstream, far away from promoter
-nondirectional -proteins bind enhancer and cause DNA to loop back around so they can bind transcription complex --> stability |
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How do enhancers regulate differential gene expression in tissues?
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-Multiple enhancers may be upstream of a given gene, all require different TF
-Different tissues have different enhancer-binding TF's --> differential gene expression |
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What is required for enhancer control of gene expression?
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Mediator Complex
allows activator protein bound to enhancer to interact with RNAP transcription complex. REQUIRED for all RNAP II promoters. |
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How are histones involved in transcriptional control?
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covalently modify histones --> reduce DNA affinity --> can be removed/moved around to expose hidden promoters, etc.
enzymes hydrolyze ATP to move histones around |
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How are histones covalently modified in DNA transcriptional control?
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-phosphorylate serine, threonine
-methylate arginine, lysine -acetylate lysine |
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Which transcription initiation factor contains a Histone Acetyl Transferase (HAT) subunit?
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TFIID (TAF I subunit)
TFIID also carries TATA Binding Protein |
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What enzyme is involved in acetylation of lysines on histones?
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Histone Acetyl Transferase (HAT)
uses Acetyl-coA as acetyl donor enhancer/activator protein recruits HAT; interaction between TFIID + enhancer protein + HAT may help TBP find its place on the gene b/c only about 1/3 of all genes have TATA box. |
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What is the approximate size of the RNAP II Holoenzyme complex?
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RNAP II 12 subunits, 600 kDa
GTF's with ~32 subunits, 1,600 kDa |
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Why are subsequent rounds of transcription usually faster than the first "pioneer" round?
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RNAP II and TFIIH leave the complex but the rest stays behind
no need to re-assemble everything, just get a new RNAP II and TFIIH |
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What is the role of RNAP II's c-terminal domain in transcription?
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-5 residue repeat x 50, lots of serines
-gets phosphorylated by CTD Kinase --> repels DNA -attracts HAT, which removes histones ahead of RNA pol -attracts proteins that remodel histones in back of RNAP/zip up the DNA |
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When does mRNA processing occur?
What happens? |
during transcription!
5' cap 3' polyA tail splicing to remove introns |
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How is the 5' cap added to mRNA's?
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CAPPING ENZYME
-binds RNA pol CTD -hydrolyzes 5' gamma-phosphate -attaches 7-METHYLGUANOSINE to remaining diphosphate via a 5' to 5' linkage (G-P to nt-P-P --> G-P-P-P-nt) |
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How is the poly-A tail added to mRNA?
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POLY-A POLYMERASE associated w/RNAP CTD
-no template required -looks for conserved AAUAAA @ end of mRNA near stop codon, adds polyA tail there. |
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What is the benefit of having introns and exons?
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differential splicing --> one gene can be spliced to make different proteins
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What is the difference between introns + exons?
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introns = junk. |GU-xxxxxxx-AG| sequence
exons = actual coding sequence |
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What is heterogeneous nuclear mRNA?
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primary transcript.
called hnRNA because they're all found in the nucleus at different stages of processing and are thus heterogeneous |
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Describe the Lariat Mechanism of splicing.
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O-P-O-|GU - - - - - - A* - - - - - AG|
1. 2'-OH of A residue in center of intron attacks phosphodiester on 5' splice site; displaces it 2. new phosphodiester bond between 2' -OH on A + 5' -OH on G 3. exon 1 displaces intron and joins to exon 2. |
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What enzyme splices mRNA?
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spliceosome: Small Nuclear RibonucleoProtein, snRNP
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What is the rate of ribosome synthesis in a cell?
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~1000/minute
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How much cell energy is directed toward protein synthesis?
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~90%
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What was the major evidence for complementarity between DNA + mRNA?
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high T(m) of DNA + mRNA heteroduplex
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What is special about the TΨC arm of the tRNA?
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has pseudoureidine residue (post-transcriptional modification)
+ methylated guanine base |
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What is the basis of wobble base pairing?
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Anticodon 3'--3--2--1--5'
Codon 5'--1--2--3--3' Codon base 3, Anticodon base 1 = wobble base often due to U, G, or Inosine in anticodon base 1 |
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What are the wobble base pairs?
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Anticodon base 1 = G
Codon base 3 = C or U AC1 = U C3 = A or G AC1 = I (Inosine) C3 = A or U or C |
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What is the difference between type I and type II tRNA synthetases?
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both attach AA to tRNA's 3' adenine 3'-OH via an ESTER BOND
type I transfers to 2'-OH first, then moves to 3'-OH spontaneously type II transfers directly to 3'-OH. |
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How are amino acids activated for attachment to tRNA's?
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carboxyl group attacks ATP --> AA-AMP + PPi
PPi --> 2 Pi 2 ATP equiv consumed. |
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What are the three potential sources of error in translation?
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1. tRNA synthetase (RS) selects wrong AA
2. RS selects wrong tRNA 3. ribosome matches wrong tRNA-AA to codon on mRNA |
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What is the purpose of IF-3 in bacterial translational initiation?
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-binds E site on small subunit
-prevents large subunit from prematurely binding before start codon is positioned properly |
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What is the function of Ef:G?
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Moves into the A site of the ribosome after peptidyl transfer
Hydrolyzes GTP to shove ribosome 1 codon down the mRNA. |
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How is translation termination accomplished?
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-RF1 or 2 binds stop codon
-RF3 enters A site, uses GTP to do peptidyl transfer to WATER MOLECULE -RF1/2 released -RRF + Ef-G bind --> release mRNA |
