Why does RNA Pol need help from CAP to bind to the lac operon promoter?
Plac is a poor match to the consensus -35 and -10 promoter elements, RNA Pol cannot efficiently recognize them on its own
Together cAMP-activated CAP and RNA Pol cooperatively bind to the lac operon, increasing transcription 20-40 fold!
Is the promoter accessed by RNA Pol via the same methods in eukaryotic and prokaryotic trancription?
No
In prokaryotic, you need the sigma factor to help bind to the promoter
In Eukaryotes, RNA polymerase can freely access its site on the promoter
What is the regulative "ground state" of genes in eukaryotes?
In eukaryotes, promoters are generally inactive in the absence of regulatory proteins, so the ground state is restrictive
In eukaryotes, are genes: positively/negatively/constituitivly regulated?
Positively regulated
Initiation of transcription in eukaryotes almost always depends on the action of multiple activator proteins
What restricts access to eukaryotic promoters?
chromatin
How is transcription and translation separated in eukarotes?
In eukaryotes, transcription [in the nucleus] is separated both in space and time from translation [in the cytoplasm] giving more room for the tight regulation of gene expression at post-transcriptional level
How many RNA polymerases do animals have?
three
How many RNA polymerases do plants have?
five
How many RNA polymerases do bacteria have?
one
Eukaryotic RNA polymerases: RNAP I
RNAP I makes rRNA precursors of most ribosomal genes (28S, 18S, and 5.8S rRNA); functions in the nucleolus
Eukaryotic RNA polymerases: RNAP II
RNAP II synthesizes all mRNA precursors, miRNA and some snRNAs; functions in the nucleoplasm
Eukaryotic RNA polymerases: RNAP III
RNAP III transcribes all tRNA genes, 5S rRNA gene and some snRNAs; functions both in the nucleolus and in the nucleoplasm
Eukaryotic RNA polymerases: RNAP IV and V
RNAP IV and V (plants) synthesize siRNA that are then converted to dsRNA by an RNA-dependent RNA Pol
Eukaryotic RNA polymerases: Plastidic
Plastidic (plants) and mitochondrial RNAPs synthesize all plastidic and mitochondrial transcripts, respectively
Describe the sensitivity to α-amanitin that the different RNAPs have
RNA polymerase I is insensitive
RNA polymerase II is highly sensitive (inhibited at 1μg/ml)
RNA polymerase III is moderately sensitive (inhibited at 10μg/ml)
RNA polymerase IV is slightly sensitive (inhibited at 50μg/ml)
Who crystallized RNAPII from yeast in 2001?
Roger Kornberg, and he won a nobel prize for it!
Which RNA Polymerase's machinery is the best studied?
RNAP II-associated transcriptional machinery is the most complex and, perhaps, the best studied of all RNAPs to date
Subunit composition of RNAP II in yeast
12 subunits, classified into core/common/nonessential groups
analog of β and β' in eukaryotic RNAP II:
RPB1 and RPB2
analog of the two α subunits in eukaryotic RNAP II:
RPB3 and RPB11
analog of the ω subunit in eukaryotic RNAP II:
RPB6
In eukaryotic RNAP II, what subunits form the pincers (or jaws)?
RPB1
RPB5
RPB9
These grip dsDNA ahead of polymerase
In eukaryotic RNAP II, what subunits form the clamp?
RPB1
RPB2
RPB6
these lock polymperase onto the template near the catalytic center
it is functionally similar to the domains of DNA polymerase
How does the structure of RNAPII differ from its free conformation vs when it is engaged in elongation?
The clamp is closed
subuinits(1,2,6)
Closed clamp ensures that transcription is processive, i.e. RNAPII is able to transcribe the whole gene without falling off and terminating prematurely
What is the purpose of Mg2+ in the active site of RNA Pol II?
Mg2+ ions serve as
cofactors for these enzymes, facilitating the catalytic activity of the
polymerase. They help stabilize the negatively charged phosphate groups
in the nucleotide triphosphates (NTPs) and the DNA/RNA template,
allowing the polymerase to bind and incorporate the NTPs into the
growing nucleic acid chain.
Describe the purpose of the different elemnts of RNA Pol II: "Fork"
The “Fork” loops are positioned to open the DNA to form the transcription bubble
Describe the purpose of the different elemnts of RNA Pol II: "Pore 1"
The “Pore 1” at the bottom of the “Funnel” serves to admit NTPs to the active site and to enable extrusion of nascent RNA if RNAP backtracks during proofreading
Describe the purpose of the different elements of RNA Pol II: "Wall"
The “Wall” forces DNA to bend
Describe the purpose of the different elements of RNA Pol II: "Rudder"
The “Rudder” destabilizes the RNA-DNA hybrid after 1 turn and helps RNA exit
Describe the purpose of the different elements of RNA Pol II: "Bridge"
The “Bridge” helix plays a role in RNAP translocation: by alternating between a straight and a bent conformation it pushes the 3’ paired base of the RNA from the +1 to the -1 position
Describe the purpose of the different elements of RNA Pol II: "Lid"
The “Lid” maintains dissociation of the RNA-DNA hybrid
Describe the purpose of the different elements of RNA Pol II: "Zipper"
The “Zipper” promotes re-association of the DNA at the other end of the bubble
What is an RNAII specific inhibitor, and where does it bind?
α-amanitin, binds in "the funnel"
How does the RNAP II specific inhibitor α-amanitin behave?
α-amanitin binding impedes the conformational change of the bridge helix required for the RNAP II translocation
CTD
carboxyl terminal domain
What occurs on the C-terminus of RNAP II subunit Rpb1?
a stretch of repeated amino acids YSPTSPS
52x in humans
sites of reversible phosphorylation in the CTD of RNAP II Rpb1
YSPTSPS
Ser2, Ser5 (also maybe Ser7 and Thr4)
When does phospohorylation of the CTD of Rpb1 occur?
Transcription elongation
Phosphorylation occurs during transcription elongation as the RNAP II clears (i.e., leaves) the promoter
Describe when the different subunits of Rpb1's CTD occur: (RNAPIIa and RNAP110)
CTD is essential for life!
T/F
True
The CTD is essential for life: cells containing truncated versions of CTD (from which two-thirds or more of the repeats have been removed) are inviable!
