Transcript Transcription

Chapter 6
• How Cells Read the
Genome: From DNA to
Protein
Test Your Knowledge
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?
Video overview of transcription
“The Protein Players” - RNA polymerases, transcription factors, initiation
factors, enhancers, repressors
Prokaryotes?
Prokaryotic transcription video
Prokaryotic Transcription
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
• 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
Gene Regulatory Networks – control the number and type of
transcripts made by a cell.
video
Simple core
promoter
UAS = upstream activator sequence RE = regulatory elements
DPE = downstream promoter elements
INR = initiator sequence
Sequences that can act as promoters (TATA is preferred)
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
Transcription Factors Have Common DNA Binding Motifs
• Zinc finger
• Helix-turn-helix
• Leucine zipper
RNA Polymerase
Recognizes and binds to TATA box; TBP +
10 TBP associated factors; position set
TBP bends DNA ~80o and
forces open the minor
groove.
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)
-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,
and TFIID + TFIIA stays at TATA.
IIA
IIB
TATA
IIF
Pol IIa
IIH
Inr
initiation complex, DNA melted at Inr
Activated PIC
Transcription 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
RNA polymerase is also assisted by DNA supercoiling
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
Elongation is Coupled to RNA Processing
• Capping
• Splicing
• Polyadenylation
RNA Capping enzymes:
• Phosphatase
• Guanyl transferase – GMP in 5’ to5’
linkage
• methyltransferase
Video of transcription and capping
CBC – cap binding complex proteins also associate and protect the cap;
Later they will direct transcript in its exit from the nucleus
RNA Splicing
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
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
the lariat debranches.
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.
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.
Accurate……….
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.
……….But Flexible
Changes in splicing patterns caused by random mutations have been
an important pathway in the evolution of genes.
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
CPSF = Cleavage and
Polyadenylation Specificity Factor
CstF = Cleavage Stimulation Factor
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
Only very “select” RNAs can
be transported out of the
nucleus
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
•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
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?
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
?
A different aminoacyl-tRNA synthetase enzyme for each amino acid
Large subunit does????
Small subunit does ?????
Translation Initiation
This is the only tRNA that can bind to the small
ribosomal subunit by itself
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
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
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.
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
Proteins Fold as They are Translated on the
Ribosome
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
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.
Proteins are marked for destruction
by the addition of a small molecule
called ubiquitin on exposed lysine
residues
Cells can also regulate protein degradation by activating new
ubiquitin ligases via different mechanisms.
Proteins generally “hide” degradation signals so that they are not
active. However, there are several mechanisms for exposing the
degradation signals.
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?
Radioactivity in hemoglobin
C.Would you expect the polyribosomes to disappear if you added cycloheximide at the same
time as edeine?
control
Add inhibitor
edeine
cycloheximide
0
2
4
Time (min)
6
8