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Lecture 10

Lecture 10

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 September 28 2012 Lecture 10 Dr. Lasko Transcription elongation by Pol II is helped along by a series of elongation factors:  Different classes of elongation factors have been identified in recent years, some are direct while others are indirect  Factors which act on naked DNA (i.e. directly on DNA in a test tube without any associated proteins): o Factors which overcome sequence-dependent or drug-induced arrest of Pol II (e.g. TFIIS and PTEFb) o Factors which can boost the rate of mRNA synthesis from a DNA template (e.g. TFIIF, Elongins, ELL proteins)  Factors which regulate the rate of elongation through chromatin (no effect on transcription on DNA alone) o E.g. FACT and Elongator TFIIS, often called IIS, is a ubiquitous protein found in all eukaryotes from yeast to man; there are also some bacterial counterparts called GreA and B  Pol II has a tendency to pause or completely arrest at certain DNA sequences, followed by a backtrack which leaves the 3’OH of the nascent mRNA out of position relative to the active site o RNA Pol II will now be trying to add a nucleotide where RNA is already present  TFIIS helps Pol II overcome this by stimulating a latent Pol II endonuclease activity and creating a new 3’OH in the mRNA which is positioned correctly in the active site  Pol II can then “take another run” at the blockage; these sequences often don’t always stop Pol II, so the second run will more often than not be successful (if not, TFIIS will just try again)  The positions of conserved acidic residues in TFIIS and GreB are indicated by green circles in the diagram above o The nascent transcript is shown in red, and the DNA template is blue o The presumed locations of backtracked RNAs are indicated by the dashed arrows  TFIIS can make a contact with the RNA where Pol II has backtracked in a position where it can activate Pol II’s endonuclease activity and cut off the excess RNA o The excess RNA (1-3 nucleotides in a pause, up to 14 1 th BIOL 300 September 28 2012 Lecture 10 Dr. Lasko in a complete arrest) is degraded  GreB in bacteria is a similar protein which has a slightly less complex structure: o GreB will also catalyze cleavage of the extra RNA  Therefore, TFIIS is a protein which favors elongation which enables RNA Pol II to get through a transcriptional pause site P-TEFb (Positive transcription Elongation factor b) is a cyclin dependent protein kinase, composed of cyclin T and Cdk9 (cycling dependent kinase) subunits  Pol II routinely pauses during elongation, once approximately 30 nucleotides have been polymerized  This allows 5’ capping of the transcript (directed by the Pol II CTD) to take place o This happens BEFORE the mRNA is fully transcribed and this is allowed to happen because of this pausing mechanism  Pausing is induced by the binding of 2 proteins which are negative regulators of transcription, DSIF and NELF, to the Pol II complex  P-TEFb overcomes the pausing induced by DSIF and NELF by a mechanism involving phosphorylation of DSIF and serine 2 residues of Pol II CTD (of the 7 nucleotide repeat) o Phosphorylation of DSIF causes it to dissociate from Pol II, this stopping the pause o Remember, the more phosphorylation on the CTD, the more likely transcription is to occur  The steps in phosphorylation of target proteins by a cyclin dependant CDK complex: o First, a cyclin and a CDK subunit bind to form an active CDK complex o Then, the target protein binds to the cyclin part of the complex, placing the target phosphorylation sites in proximity to the active site of CDK o The target protein is then phosphorylated, it is then no longer able to bind cyclin and is released form the complex  Note, for ALL CDK complexes, the target protein is only able to bind once the CDK-cyclin complex is formed; it will not bind to each protein separately Negative regulators of Elongation:  DSIF: DRB Sensitivity Inducing Factor: o DRB is a drug which inhibits sensitivity of protein kinases o Heterodimer, expressed ubiquitously in eukaryotes  NELF: Negative Elongation Factor o Multi-protein complex, all 5 subunits are essential for its activity o Acts after DSIF, so once DSIF is phosphorylated and released, so is 2 th BIOL 300 September 28 2012 Lecture 10 Dr. Lasko NELF  Note: Because pausing of Pol II is transient and not normally detected, drugs like DRB (dichloro-ribofuranosyl-benzimidazole) which are protein kinase inhibitors can disable the function of P-TEFb, allowing the actions of DSIF and NELF to be revealed by detection o This is because while DSIF and NELF normally only cause pauses because of P-TEFb, they can now cause Pol II to completely arrest with P-TEFb out of the way o DSIF is still required for pausing, with or without the presence of DRB  If DRB is used in the absence of DFIS, it will not work because pausing cannot be induced (this is how DSIF got its name) o There is a protein produced in cells which regulated elongation through a method which is similar to the actions of DRB  In primordial germ cells in a the fruit fly embryo, transcription is normally repressed until later in embryogenesis  This is regulated by PG6, which acts to inhibit P-TEFb just like DRB (we don’t need to know this for the test) P-TEFb also works through phosphorylating serine 2 of the Pol II CTD:  The CTD contains a heptapeptide repeat in the Rpb 1 subunit of the CTD: YSPTSPS o Serines are at positions 2, 5 and 7, all targets of protein kinases  Serines 2 and 5 are really the only ones to be heavily phosphorylated o Phosphorylation of Ser5 predominates in the PIC (pre-initiation complex) when Pol II is moving slowly for the first few nucleotides o Phosphorylation of Ser2 predominates in the elongating Pol II complexes when Pol II is moving at full speed  Inhibition of P-TEFb by DRB preferentially inhibits phosphorylation of serine 2, suggesting that P-TEFb preferentially acts on Ser2 within the heptapeptide repeat sequence of Pol II’s CTD Factors which increase polymerase activity on naked DNA templates (TFIIF, Elongins and ELL proteins):  mRNA synthesis in vivo approaches rates of 2000 nucleotides per minute  mRNA synthesis by purified Pol II on naked DNA in vitro only reached rates of 100-
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