Download Cell and Molecular Biology

Cell and Molecular Biology
DNA Transcription
Behrouz Mahmoudi
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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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Prokaryotic Transcription
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DNA Sequences Important to Transcription
Prokaryotes
• Promoter –
•Pribnow Box (also called the -10 element) – TATAAT
•-35 element - TTGACA
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
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downstream promoter elements
INR = initiator sequence
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DPE =
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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RNA Polymerase
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Recognizes and binds to TATA box; TBP + 10 TBP
associated factors; position set
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TBP bends DNA ~80o and
forces open the minor groove.
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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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-30
+1
TATA
Inr
TAFs
Sequential
Binding
Model for
assembly of
preinitiation
complex
}
TBP
TFIID
or TBP
IIA
IIB
Eukaryotic RNA
polymerase II
IIF
Pol IIa
CTD of large subunit of Pol II
IIE
helicase
IIH
protein kinase
IIF
IIE
IIA
IIB
TATA
Inr
Pol IIa
IIH
preinitiation complex
= PIC
ATP hydrolysis
IIE
Polymerization of 1st few NTPs and
phosphorylation of CTD leads to
promoter clearance. TFIIB, TFIIE and
TFIIH dissociate, PolII+IIF elongates,
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TFIID + TFIIA stays at TATA.
IIA
IIB
TATA
IIF
Pol IIa
IIH
Inr
initiation complex, DNA melted at Inr
Activated PIC
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Transcription initiation in the cell often requires the local recruitment of chromatinmodifying enzymes, including chromatin remodeling complexes and histone acetylases greater
accessibility to the DNA present in chromatin
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RNA polymerase is also assisted by DNA supercoiling
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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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Video of transcription and capping
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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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RNA Splicing
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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
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R=A
or G,Y=C or U
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The Spliceosome Forms
snRNAs (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
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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
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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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Poly A polymerase – no
template strand required
All of the A nucleotides are
derived from ATP
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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Large
subunit does????
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Small subunit does ?????
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Translation Initiation
This 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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Proteins Fold as They are Translated on the
Ribosome
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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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Cap hydrolyses ATP; regulates
entry
Proteolytic core
Cap hydrolyses
ATP; regulates exit
Proteasomes are a major mechanism by which cells regulate the
concentration of particular proteins and degrade misfolded proteins.
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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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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 edeineinhibited 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?
Radioactivity in hemoglobin
C.Would you expect the polyribosomes to disappear if you added cycloheximide at the same
time as edeine?
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control
Add inhibitor
edeine
cycloheximide
0
2
4
Time (min)
6
8
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