Week of FEB-22

M T W R F
10-11 10-11
Iryna Run
office hours office hours
LLAB 114 LLAB 114
NEW!
11:00-11:50 noon-1:00 11:00-11:50 11:00-11:50
Class GG Class Class
LLAB 10 office hours LLAB 10 LLAB 10
Questions? LL114
LLAB 319
12-1
Steve
office hours
LLAB 114
6:30-8:30 5:15-6:15 4-5
Recitation GG GG
LLAB 10 office hours office hours
LLAB 319 LLAB 319
1
221.S16
 Week of FEB-29

M T W R F
10-11 10-11
Iryna Run
office hours office hours
LLAB 114 LLAB 114
11:00-11:50 noon-1:00 11:00-11:50 11:00-11:50
Class GG Class Class
LLAB 10 office hours LLAB 10 LLAB 10
Questions? LL114
LLAB 319
12-1
Steve
office hours
LLAB 114
6:30-8:30 5:15-6:15
Recitation GG
LLAB 10 office hours
molecular LLAB 319
techniques
2
221.S16
 Week of MAR-7

Spring Break

3
221.S16
 Week of MAR-14

M T W R F
10-11 10-11
Iryna Run
office hours office hours
LLAB 114 LLAB 114
11:00-11:50 noon-1:00 11:00-11:50 11:00-11:50
Class GG Class Class
LLAB 10 office hours LLAB 10 LLAB 10
Questions? LL114
LLAB 319
12-1
Steve
office hours
LLAB 114
5-6:30 6:30-8:00 6:30-8:30 5:15-6:15
Review Review Exam-2 GG
LLAB 10 LLAB 10 LLAB 10 office hours
LLAB 319

4
221.S16
 Prokaryotic transcription: factor-independent termination

5
factor-independent (intrinsic)
221 S16
 Prokaryotic transcription: factor-independent termination

Bacterial
plasmid

Control Experimental
PBAD: Promoter from Arabinose operon
RBS: Ribosome Binding Site T : Terminator (experimental)
T : Terminator T : Terminator (always present)
GFP: Green Fluorescent Protein
RFP: Red Fluorescent Protein
ColE1: Ori from high copy number plasmid
ampR: Ampicillin Resistance Gene
6
araC: repressor factor-independent (intrinsic)
221 S16
 Prokaryotic transcription: factor-independent termination

7
factor-independent (intrinsic)
221 S16
 Automated synthetic oligonucleotide synthesis

5’-TCTTAGCCGATCGATCGATCGATCGATCGATCGATCGA-3’
3’-AGAATCGGCTAGCTAGCTAGCTAGCTAGATCGATCGAT-5’

8
221 S16
 Prokaryotic transcription: factor-independent termination

Bacterial
plasmid

Control Experimental
PBAD: Promoter from Arabinose operon
RBS: Ribosome Binding Site T : Terminator (experimental)
T : Terminator T : Terminator (always present)
GFP: Green Fluorescent Protein
RFP: Red Fluorescent Protein
ColE1: Ori from high copy number plasmid
ampR: Ampicillin Resistance Gene
9
araC: repressor factor-independent (intrinsic)
221 S16
 Prokaryotic transcription: factor-independent termination

10
221 S16 Nature Meth 10: 659-664(2013)
Prokaryotic transcription: factor-independent termination

3’
U U UU
U
C C
C G U
U CG GG U UU
U GC
C G
0 0.2

TS = 24

3’
G U CU
C
C C
C G U
U CG GG U UU
U GC
C G
TS = 10
0 0.2

11
http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi
 Prokaryotic transcription: factor-independent termination

12
factor-independent (intrinsic)
221 S16
 Prokaryotic transcription: factor-independent termination

13
221 S16 Nature Meth 10: 659-664(2013)
 Prokaryotic transcription:
factor-dependent termination
RNA
Rho–dependent termination
5’

Rho binds to “Rho-

utilization”, or “Rut” sites
located near the 5’ end of an RNA
RNA transcript, then 5’
translocates along RNA
molecule to the transcription
bubble, where it interacts
with RNAP (not shown here)
and displaces RNA from the
template… RNA
5’

RNA 14
221 S16 5’ Banerjee et al., (2006) 44:11 B. Keith
 Eukaryotic transcription
familiar themes, increased complexity

15
221 S16 B. Keith
 Eukaryotic RNA polymerases

Polymerase RNA products

RNA Polymerase I rRNA
RNA Polymerase II mRNA, snRNA, miRNA
RNA Polymerase III tRNA, snRNA

16
221 S16
 Eukaryotic RNA polymerase structure

Species Enzyme Subunits

E. coli RNA polymerase 5 (holoenzyme)

human RNA polymerase I 14

4 subunits
human RNA polymerase II 12 in common
human RNA polymerase III 16

17
221 S16 Cramer et al., Science 288:640 (2000)
 Eukaryotic RNA polymerase II

Crystal structures of RNA polymerase have been solved

describing different functional complexes with
DNA, RNA, nucleotides or other proteins.

18(2000)
Cramer et al., Science 288:640
221 S16
 Evolutionary conservation of RNAP structure

19
221 S16 Nature Reviews Microbiology 11, 713–719 (2013)
 RNA polymerase initiation

Prokaryotic RNA polymerase

Eukaryotic RNA polymerase II

20
Nature Reviews Molecular Cell Biology 5: 403-410 (2004)
221 S16
 Eukaryotic RNA polymerase II:

• preinitiation complex (PIC) formation (general transcription factors)

• initiation & elongation

• co-transcriptional RNA processing

• 5’ capping
• mRNA splicing
• termination (cleavage and polyadenylation)

21
221 S16
 Eukaryotic RNA polymerase II: preinitiation complex formation
General Transcription Factors (GTFs)

TFIID (15 proteins) including

TATA-binding protein (TBP)

TFII”x”: Transcription Factor for Pol

II
RNAPII

RNA polymerase II is promoter “blind” 22

221 S16 Griffiths et al. (2012) F8-12
 Eukaryotic RNA polymerase II: initiation & elongation

RNAPII

RNA polymerase II is promoter “blind”

RNAPII

CTD: C-terminal Domain of large subunit

23
221 S16
 Eukaryotic RNA polymerase II transcription

Meinhart & Cramer (2004) 430, 223-226

CTD: C-terminal Domain of large subunit

Tyr-Ser-Pro-Thr-Ser-Pro-Ser CTD is phosphorylated on specific

Ser residues…
52 copies (human)
24
221 S16
 RNA processing enzymes are recruited
by the CTD tail of the large subunit of RNA
polymerase II

Differential phosphorylation of CTD repeats

helps recruit specific factors that modify
eukaryotic pre-mRNAs to make them
functional mRNAs. Specifically,

5’ capping
splicing
polyadenylation (termination)

25
221 S16 B. Keith