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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
TATA TAFs

+1
Inr or TBP

Sequential Binding Model for assembly of preinitiation complex

TBP

}
Pol IIa

TFIID

IIA IIB
Eukaryotic RNA polymerase II

IIF

CTD of large subunit of Pol II helicase IIH protein kinase

IIE

IIE IIB
TATA

IIF

IIA
Inr

Pol IIa IIH preinitiation complex

ATP hydrolysis

= PIC
IIE IIF

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
TATA

IIA

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 couldnt 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 dont fold properly after being translated?

Transfer RNA

anticodon- 3 to 5 sequence that matches the complementary 5 to 3sequence (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 ?????

This is the only tRNA that can bind to the small ribosomal subunit by itself

Translation Initiation

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? C.Would you expect the polyribosomes to disappear if you added cycloheximide at the same time as edeine? Radioactivity in hemoglobin

Add inhibitor

control

edeine cycloheximide

4
Time (min)