www.soran.edu.iq cell and molecular biology behrouz mahmoudi dna transcription 1
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Cell and Molecular Biology
Behrouz Mahmoudi
DNA Transcription
1
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1. What are two major differences between transcription in prokaryotic and eukaryotic cells?
2. RNA polymerase and DNA polymerase enzymes catalyze the “same” reaction, but there are some distinct differences in what is required to make them begin catalysis and end catalysis. What are these differences?
3. Which is more accurate, DNA replication or RNA transcription?
4. Explain the proteins and mechanisms involved in the initiation of transcription
5. What determines how many copies of a transcript (mRNA) are made?
6. How are elongation and termination of the transcript regulated?
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“The Protein Players” - RNA polymerases, transcription factors, initiation factors, enhancers, repressors
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Prokaryotes• Promoter –
• Pribnow Box (also called the -10 element) – TATAAT• -35 element - TTGACA
DNA Sequences Important to Transcription
Eukaryotes• Promoter –(asymmetrical sequence)
• Basic core promoter –TATA box (TATAAA(A)); within 50bp upstream of start site; found in unicellular eukaryotes
• Core promoter PLUS• Downstream promoters• Proximal promoter elements
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• Initiator sequences
• Regulatory Elements/Response Element - Response elements are the recognition sites of certain transcription factors Most of them are located within 1 kb from the transcriptional start site.
• Enhancer elements -upon binding with transcription factors (activators),
can enhance transcription; located either upstream or downstream of the transcriptional initiation site.
• Upstream enhancer elements• Downstream enhancers• Distal enhancer elements
• Silencers - upon binding with transcription factors (repressors), can repress transcription.
• Insulators
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Gene Regulatory Networks – control the number and type of transcripts made by a cell.
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Simple core promoter
UAS = upstream activator sequence RE = regulatory elements INR = initiator sequence DPE = downstream promoter elements
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Sequences that can act as promoters (TATA is preferred)
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Proteins Involved in Transcription
RNA Polymerase
General (or Basal) Transcription Factors:
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
Transcription Factors that Bind to Regulatory Elements
Holoenzyme or
Initiation Complex
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Transcription Factors Have Common DNA Binding Motifs
• Zinc finger
• Helix-turn-helix
• Leucine zipper
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Recognizes and binds to TATA box; TBP + 10 TBP associated factors; position set
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Recognizes and binds to TATA box; TBP + 10 TBP associated factors
Binds and stabilizes the TFIID complex
Recruits RNA pol II + TFIIF to the location
Two subunits - RAP38 & RAP74. Rap74 has a helicase activity; RAP38 binds RNAPolII
Two subunits - recruits TFIIH to the complex thereby priming the initiation complex for promoter clearance and elongation
complex of 9 subunits. One w/ kinase activity; one w/ helicase activity; one is a cyclin (cdk7)
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Sequential Binding
Model for assembly of preinitiation
complex
-30 +1
TATA Inr
Polymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance. TFIIB, TFIIE and TFIIH dissociate, PolII+IIF elongates, and TFIID + TFIIA stays at TATA.
IIB
Eukaryotic RNA polymerase II
TFIID}TBP
TAFs
IIB
IIECTD of large subunit of Pol II
Pol IIa
or TBP
IIA
IIF
helicase
protein kinase
IIH
TATA Inr
IIA Pol IIa
IIFIIE
preinitiation complex
TATA Inr
IIAIIB Pol IIa
IIFIIE
ATP hydrolysis
initiation complex, DNA melted at Inr
IIH
IIH
= PIC
Activated PIC
www.soran.edu.iq 20Transcription initiation in the cell often requires the local recruitment of chromatin-modifying enzymes, including chromatin remodeling complexes and histone acetylases - greater accessibility to the DNA present in chromatin
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Phosphorylation of the carboxy terminal domain (CTD) of one of the subunits of RNA PolII;
RNA polymerase II dissociates from the transcription factors and other protein complexes that were required
for assembly and elongation begins
Phosphorylation also promotes the accumulation of elongation factors – other proteins that arrest transcription long enough to recruiting RNA processing enzymes
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Elongation is Coupled to RNA Processing
• Capping• Splicing• Polyadenylation
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RNA Capping enzymes:• Phosphatase
• Guanyl transferase – GMP in 5’ to5’ linkage
• methyltransferase
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CBC – cap binding complex proteins also associate and protect the cap;
Later they will direct transcript in its exit from the nucleus
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How Introns Are Identified:
Consensus sequences at (5’ to 3’ direction)
•5’ splice site
•Lariate loop closure site of the intron sequence
•3’ splice site
R=A or G,Y=C or U
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The Spliceosome FormssnRNAs (U1, U2, U4, U5 and U6) and associated proteins =
snRNPs
• U1 binds to the GU sequence at the 5' splice site, along with accessory proteins/enzymes,
• U2 binds to the branch site, and ATP is hydrolyzed;
• U5/U4/U6 trimer binds, and the U5 binds exons at the 5' site, with U6 binding to U2;
• U1 is released, U5 shifts from exon to intron and the U6 binds at the 5' splice site;
• U4 is released, U6/U2 catalyzes transesterification, U5 binds exon at 3' splice site, and the 5' site is cleaved, resulting in the formation of the lariat;
• U2/U5/U6 remain bound to the lariat, and the 3' site is cleaved and exons are ligated using ATP hydrolysis. The spliced RNA is released and the lariat debranches.
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Rearrangements that occur during splicing
• U1 replaced by U6
• BBP (branch binding protein) and U2
• U5 complex branch forming enzymes in U6 and U2
Allows for “check and recheck” at each splice site.
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Why is splicing so accurate?
Introns are small-large;
Exons are about 150bp long
Exons might be easily identified, while introns probably couldn’t be.
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As the RNA is being transcribed, SR proteins (rich in serine (S) and arginine (R)) sit down on the exons. Along with the U proteins, demarcates the start and end of the exon.
Capping proteins or polyA binding proteins act as markers at either end of the transcript.
Other hnRNPs (heterogeneous nuclear RNPs) bind along the introns, helping to distinguish these sequences from exons.
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Changes in splicing patterns caused by random mutations have been an important pathway in the evolution of genes.
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3’ end splicing sequence
• AAUAAA
• Cleavage site CA – 10-30 nucleotides downstream
• Polyadenylation site – GU- or U-rich region about 30 nucleotides downstream from the cleavage site
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CPSF = Cleavage and Polyadenylation Specificity Factor
CstF = Cleavage Stimulation Factor
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All of the A nucleotides are derived from ATP
Poly A polymerase – no template strand required
Poly A binding proteins remain until mRNA undergoes translation
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Only very “select” RNAs can be transported out of the nucleus
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Guided diffusion along the FG-repeats displayed by nucleoporins
Proteins bound to mature mRNA molecules and that signal completed splicing have nuclear export signals as a part of their sequence
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•hNRPs “straighten out” the mature mRNA so that nuclear export signals can be “read”
•5’ cap enters the pore first
•Many of the RNA binding proteins fall off as mRNA exits the nucleus
•Initiation factors (elF-4G and elF-4E) immediately bind to the 5’ capping complex (which falls off) and to the polyA tail, forming a loop
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Test Your Knowledge – Translation
• Transcription requires only changing a DNA code of nucleotides into a similar RNA code of nucleotides, while translation involves changing the RNA code into what?
• What are codons and what “reads” codons?
• What is “wobble” and how is it related to translation?
• What attaches amino acids to t-RNA?
• What are the “parts” of the ribosome? What function does each part perform?
• What are the A, P, and E sites of a ribosome? What binds at each of these sites?
• Does anything beside the ribosome participate in elongation of the amino acid chain? If so, what is it and what does it do?
• What signals where translation starts and stops?
• What happens to improperly translated or proteins that don’t fold properly after being translated?
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Transfer RNA
• anticodon- 3’ to 5’ sequence that matches the complementary 5’ to 3’sequence (codon) on the mRNA
• Acceptor arm - Amino acid code on 3’ end
• T and D loops – provide structure for interface with aminoacyl-tRNA synthetase
?
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A different aminoacyl-tRNA synthetase enzyme for each amino acid
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Translation InitiationThis is the only tRNA that can bind to the small ribosomal subunit by itself
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Protein made in 5’ to 3’ direction, with N-terminal end made first
General Mechanism
• A site is where new codon is translated
• P site is where the growing peptide chain is kept and new aa are attached
• E site is where “naked” t RNA exit the ribosome
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More Detailed View
New tRNA carrying amino acids are accompanied by elongation factor called EF-Tu
The tRNA-ETu occupies a hybrid binding site (not quite in A)
Correct codon-anticodon pairing triggers ETu to split GTP and fall off, and tRNA moves into the A position
The delay caused by the association/dissociation of ETu helps increase accuracy of translation
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Elongation factor G (EF-G) then binds near the A site, forcing the tRNAs containing the new amino acid and the growing chain into the next (P and E) sites on the ribosome
EF-G splits GTP, changes conformation and falls off, thus increasing the speed of translation.
GTP exchange factors continually recharge the GTP on both of the elongation factors.
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Stop Codons = UAA, UAG, UGA
No tRNA binds to this set of codons
One of these codons at the A site attracts a release factor
Ribosome adds a water to the last peptide, creating the carboxyl end
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Hsp 70 works to help fold early in the lifecycle of a protein
Hsp 60 works like a quality control chamber after a protein is completely folded
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Proteasomes are a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins.
Proteolytic core
Cap hydrolyses ATP; regulates exit
Cap hydrolyses ATP; regulates entry
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Proteins are marked for destruction by the addition of a small molecule called ubiquitin on exposed lysine residues
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Cells can also regulate protein degradation by activating new ubiquitin ligases via different mechanisms.
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Proteins generally “hide” degradation signals so that they are not active. However, there are several mechanisms for exposing the degradation signals.
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Time (min)
Rad
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y in h
em
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Add inhibitorcontrol
edeine
cycloheximide
You have isolated an antibiotic named edeine, from a bacterial culture. Edeine inhibits protein synthesis but has not effect on either DNA synthesis or RNA synthesis. When added to a reticulocyte lysate, edeine stops protein synthesis after a short lag, as shown below. By contrast, cycloheximide stops protein synthesis immediately. Analysis of the edeine-inhibited lysate by density-gradient centrifugation showed that no polyribosomes remained by the time protein synthesis had stopped. Instead, all the globin mRNA accumulated in an abnormal 40S peak, which contained equimolar amounts of the small ribosomal subunit and initiator tRNA.
A.What step in protein synthesis does edeine inhibit?
B.Why is there a lag between addition of edeine and cessation of protein synthesis? What determines that lag?
C.Would you expect the polyribosomes to disappear if you added cycloheximide at the same time as edeine?