Transcript Transcription Speed - UCSF Tetrad Program

Biochemistry 201
Biological Regulatory Mechanisms
January 24, 2013
Transcription elongation and its regulation
References
A few of the many insights from RNA polymerase structures
Cramer, P. (2002) Multisubunit RNA polymerases. Curr Opin Struct Biol 12:89-97.
Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the holo story. Curr Opin Struct Biol 13:31-9.
*Cramer, P. (2004) RNA polymerase II structure: from core to functional complexes. Curr Opin Genet Dev 14:218-26.
Review.
Wang, D. Bushnell DA, Westover KD, Kaplan, CD, Kornberg RD. Structural basis of transcription: role of the trigger loop
in substrate specificity and catalysis. Cell. 2006 Dec 1;127(5):941-54.
Cramer, P. (2007). Gene transcription: extending the message. Nature, 448(7150), 142-3.
*Vassylyev, DG, Vassylyeva, MN, Zhang, J, Landick, R (2007). Structural basis for substrate loading in bacterial RNA
polymerase. Nature, 448(7150), 163-8.
IV. Proofreading
*Zenkin, N, Yuzenkova, y Severinov K Transcript-assisted transcriptional proofreading.
Science. 2006 Jul 28;313(5786):518-20
Sydow JF, Cramer P. (2009) RNA polymerase fidelity and transcriptional proofreading.Curr Opin Struct Biol. 2009
Dec;19(6):732-9. Epub 2009 Nov 13.
Sydow JF, Brueckner F, Cheung AC, Damsma GE, Dengl S, Lehmann E, Vassylyev D, Cramer P.(2009) Structural basis
of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol Cell. Jun
26;34(6):710-21.
V. Pausing
Artsimovitch, I. and Landick, R (2000). Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of
signals. PNAS 97: 7090-7095
Zhang J, Palangat M, Landick R. Role of the RNA polymerase trigger loop in catalysis and pausing. Nat Struct Mol Biol. 2010
Jan;17(1):99-104. Epub 2009 Dec 6.
*Shaevitz, j. Abbondanzieri E, Landick R. and Block S (2003) Backtracking by single RNA polymerase molecules observed at near
base pair resolution. Nature 426: 684-687
Herbert, K., La Porta, A, Wong B, Mooney, R. Neuman, K. Landick, R. and Block, S.(2006). Sequence-Resolved Detection of
Pausing by Single RNA Polymerase Molecules. Cell 125:1083-1094
*Weixlbaumer, A, Leon, K, Landick, R and Darst SA (2013) Structural basis of transcriptional pausing in bacteria. Cell, in press
VI. Regulation through the 2˚ channel
Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL. (2004) DksA: a critical component of the
transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP.
Cell. 6:311-22.
Role of the RNA Pol II CTD
*McCracken, S, Fong, N, Yankulov, K, et al. (1997). The C-terminal domain of RNA polymerase II couples mRNA processing to
transcription. Nature, 385(6614), 357-61.
Tietjen,J. ……Ansari, A. Chemical-genomic dissection of the CTD code (2010) NMSB: 17: 1154-1162
Mayer, A. ….Cramer, P. Uniform transitions of the general Pol II transcription apparatus (2010) NMSB 17:1272-79
Buratowski, S (2009) progression through the RNA polymerase II CTD cycle ( Review). Mol Cell 36: 541-546
Chapman, R… Eick, D. Molecular evolution of the RNA polymerase CTD. Trends in Genetics (2008): Jun;24(6):289-96. Epub
2008 May 9. Review.PMID: 18472177
Elongation Control
BBA2013-- Issue 1874 devoted to reviews of transcription elongation
Zhou Q, Li T, Price DH (2012) RNA polymerase II elongation control .Annu Rev Biochem. 2012;81:119-43.
Rougvie A and Lis JT (1988) The RNA Polymerase II Molecule at the 5’ end of the uninduced hsp70 gene of D. melangaster is
transcriptionally engaged. Cell 54: 795-804
Zobeck, KL….Lis Jt (2010) Recruitment timing and dynamics of transcription factors at the Hsp70 Loci in Living Cells Mol Cell
40 965-75
Peterlin, BM and Price DH (2006) Controlling the Elongation Phase of transcription with P-TEFb Mol Cell 23: 297 – 305
Nechaev S…..Adelman K. (2010) Global Analysis of short RNAs reveals widespread Promoter Proximal Stalling and Arrest of Pol II
in Drosophila Science 327: 335-38
Gilchrist, DA,……Adelman, K. (2010) Pausing of RNA Polymerase II Disrupts DNA specified Nucleosome Organization to enable
precise gene regulation. Cell 143: 540-51
Chen, Y.,….Handa, H.(2009) DSIF, the Paf1 complex and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II
elongation. Genees Dev 23: 2765 -77
Liu, Y……Hahn, S. (2009) Phosphorylation of the transcription elongation factor Spt5 by yeast Bur1 kinase stimulates recruitment of
the PAF complex. MCB 29: 4852-63
Wu, C-H,….Gilmour, D. ( 2003)NELF and DSIF cause promoter proximal pausing on the Hsp70 promoter in drosophila. Genes Dev
17: 1402-14
Kim, J Guemah M and Roeder, RG.(2010) The human PAD1 Complex Acts in Chromatin Transcription elongation both
independently and cooperatively with SII (TFIIS) Cell 140: 491 -503
Genome wide elongation technologies
Churchman LS, Weissman JS. (2011) Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469:
368-73. doi: 10.1038/nature09652. (Net-seq)
Kwak, H… and Lis, JT (2013) Precise maps of RNAP reveal how promoters direct initiation and pausing. Science 339: 950 (PROseq)
Important Points
1. Cellular RNA polymerases have no structural similarities to DNA polymerases; even though they carry
out similar reactions, they are a separate evolutionary invention.
2. Cellular RNA polymerases have many moving parts. For example, incoming NTPs first base pair with
the template in a catalytically inactive form and are subsequently pushed into the active site by folding of
the “trigger loop”. This movement links correct nucleotide recognition to catalysis and thereby increases
fidelity. In other words, the polymerase takes two looks at the incoming NTP.
3. The active site of cellular RNA polymerases can be regulated by accessory proteins that penetrate the
secondary channel (also called the pore), position a Mg ion, and thereby cause the active site to cleave
RNA rather than polymerize it. This reaction is not simply the reverse of the polymerization reaction.
4. RNA proofreading occurs when a mispaired nucleotide positions a Mg at the active site, stimulating
cleavage reaction.
5. Transcriptional pauses are integral to the transcription process and are integral to transcriptional
regulation and are extensively used in both prokaryotes and eukaryotes
6. The RNA polymerase CTD is a long series of 7-amino acid repeats. When transcription is initiated,
serine 5 of the repeat is phosphorylated by TFIIH. As elongation proceeds, serine 5 is gradually
dephosphorylated and serine 2 is gradually phosphorylated by enzymes carried along with the RNA
polymerase. This dynamic pattern of modification couples transcription to processing of the newlysynthesized RNA.
7. Promoter proximal pauses ( 20-50 nucleotides) are extensively used for regulation in eukaryotes.
Replication vs transcription
Replication
Speed
500 nucs/sec: bacteria
50 nucs/sec: euks
Error rate 1/109(including
mismatch repair)
Job
Copy every sequence in
the genome once
Transcription
10-30 nucs/sec
1/104- 1/105
Transcribe segments
of the genome at
highly variable rates
RNA polymerases vs. DNA polymerases
Similarities
1. Polymerize NTPs using DNA as template
2. Similar reaction mechanism
3. Both remove errors
Differences
1. Ribonucleoside vs deoxyribonucleside triphosphates
2. No structural similarity
3. RNAP initiates de novo; DNAP elongates prexisting chains
4. RNAP active site does both NTP addition and proofreading
5. Active site of RNAP is highly regulated, enabling a
dynamic response to signals during elongation
Structure of
RNAP
Cutaway view of
elongating complex
Current view of Role of reactions at active site
X
X
(?)
Elementary Pause Complex
Current view of Role of reactions at active site
(?)
Elemental Pause Elongation Complex
X
X
Current view of Role of reactions at active site
(?)
Elemental Pause Elongation Complex
Transcript
cleavage
(1) Structure of the elongation complex
“Frozen” elongating complexes can be assembled on a nucleic acid scaffold
Complexes were used to determine RNAP structure during nucleotide addition
Determined two structures of elongating RNA polymerase
a) Elongation complex with non-hydrolyzable NTP
b) Elongation complex with non-hydrolyzable NTP
and streptolydigin ( elongation inhibitor)
RNA-P looks at each incoming NTP twice before addition
Substrate enters through 2˚ channel
NTP binds at “preinsertion site”
usingW-C base pairing; RNAP
contacts discriminate NTP /dNTP;
2nd Mg++ too far for catalysis
(structure in the presence of NTP and
streptolydigin or -amanitin)
Trigger-loop folds and forms 3-helix
bundle with bridge helix; active center
closes allowing additional check for
complementarity; 2˚ channel constricts
(structure in the presence of NTP)
Incorporation of mononucleotide and
release of pyrophosphate
Elements of a back-tracked pause
1.
Enabled by ability of RNA to translocate relative to the DNA
template; when there is a less stable DNA/RNA hybrid, tendency of
RNA is to backtrack until a more stable RNA/DNA hybrid is achieved
2. Backtrack pauses are reduced by creating a more stable RNA/DNA
hybrid, or by addition of GreA (promotes transcript cleavage and
realignment of active center
3. Position of RNA polymerase on DNA can be determined by
footprinting using exonuclease III (degrades DNA from 3’end)
(2) The Transcript Cleavage Reaction
Transcript cleavage factors bind in the 2˚ channel; a Mg++
bound to the tip mediates cleavage of a “backtracked” RNA
Misincorporated NTPs promote backtracking; transcript cleavage factors
promote error correction (cleavage factors also promote elongation)
RNAP alone can also correct errors. Here a backtracked RNA chain binds
2nd Mg++ to promote cleavage by the active site
(3) Transcriptional pauses
are really important
Coordinate transcription (RNAP movement) with:
1) Folding nascent RNA
2) Other RNA processes
translation, degradation, export, splicing
3) Regulator binding (TAR—HIV; RfaH prokaryotes)
Promoter proximal pauses poise RNAPII for gene expression
in metazoans
The central role of pausing in control
Elemental Pause Elongation Complex
(?)
Transcript
cleavage
AT rich;
misincorporation
11 nt from 3’ end
of mRNA
~ 8 nt from 3’ end; terminal nts highly
enriched in U’s; A-U bp are very weak
Elements of a back-tracked pause
1.
Enabled by ability of RNA to translocate relative to the DNA
template; when there is a less stable DNA/RNA hybrid, tendency of
RNA is to backtrack until a more stable RNA/DNA hybrid is achieved
2. Backtrack pauses are reduced by creating a more stable RNA/DNA
hybrid, or by addition of GreA (promotes transcript cleavage and
realignment of active center
3. Position of RNA polymerase on DNA can be determined by
footprinting using exonuclease III (degrades DNA from 3’end)
How to measure pauses
Time (Min)
Run-off transcript--
Pause transcript--
Pauses are characterized by duration and
“efficiency” (probability of entering the
pause state at kinetic branch between
pausing and active elongation)
Aliquots of a synchronized, radiolabeled, single-round transcription assay were removed at various times and
electrophoresed on a polyacrylamide gel; separation by size
Pauses can also be measured using single molecule technology
Pauses can also be measured genome wide using NET-seq
Matt Larson ( Weissman lab)
Regulating Termination: Attenuation control
1. Stabilizing alternative 2˚structures of mRNA can lead to either elongation or termination
2. External inputs can alter the equilibrium between mRNA states
3. RNA polymerase pausing is critical for this regulatory mechanism
Case study: Use of a Pause hairpin in “Attenuation” at the trp
operon
Low Trp
High Trp
1:2 is a pause hairpin
3:4 is an intrinsic terminator
Leader peptide has tryptophan residues
2:3 is an “antiterminator”
hairpin
Regulated “attenuation” (termination) is widespread
Switch between the “antitermination” and “termination”
Stem-loop structures can be mediated by:
1. Ribosome pausing ( reflects level of a particular charged tRNA): regulates
expression of amino acid biosynthetic operons in gram - bacteria
2. Uncharged tRNA: promotes anti-termination stem-loop in amino acyl tRNA
synthetase genes in gm + bacteria
3. Proteins: stabilize either antitermination or termination stem-loop structures
4. Small molecules: aka riboswitches
5. Alternative 2˚ structures can also alter translation, self splicing, degradation
NusG, the only universal elongation factor,
exhibits divergent interactions with other regulators
E. coli NusG: A 21kD essential elongation factor
NTD
NGN domain
CTD
KOW domain
Activities:
1. Increases elongation rate
2. suppresses backtracking
3. Required for anti-termination mechanisms
4. Enhances termination mediated by the rho-factor
How does one 21Kd protein mediate all of these activities?
NusG-like NTD binds across the cleft in all three
kingdoms of life, apparently locking the clamp
against movements (& encircling DNA)
The N-terminal NGN domain increases elongation and decreases pausing
adapted from Martinez-Rucobo et al. 2011 EMBO J. 30:1302
The CTD of NusG interacts with other protein partners
50 µM
CTD
NusE
Rho
NusE is part of a complex of proteins mediating antitermination/termination
depending on its protein partners
Rho is an RNA binding hexamer that mediates termination by
dissociating RNA from its complex with RNA polymerase and DNA
using stepwise physical forces on the RNA derived from alternating
protein conformations coupled to ATP hydrolysis
Although the CTD mediates the protein interactions involved in termination and antitermination, full length NusG is
required for both processes, presumably because NusG must be tethered to RNA polymerase for these functions
Coupled syntheses.
J W Roberts Science 2010;328:436-437
Published by AAAS
Elongation control in eukaryotes
The RNA Polymerase II CTD (or tail)
Heptad repeat unit
YSPTSPS
P
P
P
2
5
7
Proline can be cis or -trans
5 repeats in plasmodium
26 repeats in yeast
52 repeats in mammals
Regions upstream (R1) and
downstream (R3) of the heptad repeat
region are enriched in the submotifs
What is the role of the Pol II CTD?
Mouse RNA Pol II
wt
52
 - amaR
CTD
5
50 hrs.
HeLa
cells
Introduce
CTD construct
examine
RNAs
Splicing, processing of 3’
end, termination
were all affected
 - amanitin
Nature 385: 357 (1997)
How the Polymerase CTD Couples Transcription to other processes
Kinase/ phosphatase
2
5
7
YSPTSPS
TF II H,
Mediator
Factors recruited
capping factors
P
elongation
pTEFb
YSPTSPS
(Cdk9)
In S. cerevisiae, shared by Cdk1 and Bur 1
P
P
splicing components histone
methylase
DNA repair enzymes
Further
elongation
phosphatases (Rtr1(2?)
YSPTSPS
P
3’ end processing factors
Termination
Phosphatases (Fcp1, ssu72)
YSPTSPS
Mediator, activators, GTFs
TECs are community organizers
The major steps in mRNA processing (trx, 5’ capping, polyA addition, splicing) all occur together on a transcript extruded from the
exit channel of RNAP although they can be reconstituted independently in vivo
Principles of “cotranscriptionality” to integrate nuclear metabolism
1.Permits coupling between different biogenesis steps; eg crosstalk; suspected when decreasing one step has effects on 2 nd; could
always be indirect
a. Landing pad—increase concentration of reactants—proteins involved in capping etc
b. Allosteary: guanosyl transferase of capping enzyme activated by interaction with phosphorylated CTD
c. Kinetic coupling—optimize timing
2. Impose order or control
a. Juxtaposition of proteins permits assembly, competitive interactions handoffs; often mutually exclusive PPis
b. directions emanating from phopshorylation state of CTD
3. A locator for nuclear machines –DNA repair, modification etc
Bentley: Cotranscriptionality Mol cell rev 2009
Elongation control in process coupling
Fast elongation favors exon skipping whereas slow elongation
favors exon inclusion
De la mata
What don’t we know about the CTD?
1.
2.
What is the role of Ser-7 phosphorylation?
Ser-7 shows high phosphorylation across highly transcribed protein
coding genes in S. cerevisae, but no role yet ascribed to this modification
What is the significance of different markings when comparing non-coding
and protein coding genes and how is this difference set up?
3. To what extent do interdependent and co-occurrence of marks set-up
bivalent/multivalent recognition patterns
4. Genome wide ChIP analysis indicates some factors thought to be recruited by
Ser-2 phosphorylation appear either signficantly prior to or after that event.
Explanation?
See Tietjen…..Ansari NMSB(2010) 17: 1154
Mayer….Cramer NMSB (2010): 1272
NusG orthologue Spt5 functions with Spt4 ( and other proteins) in
elongation control
Spt5: essential in yeast
A promoter proximal pause is characteristic of transcription of many genes in higher
eukaryotes
Characteristics of paused polymerase ( pioneering work by
John Lis Hsp70 locus in Drosophila)
1.In open complex ( KMnO4 footprinting)
2.Some fraction can elongate (nuclear run-on experiments)
Later work:
3. Ser-5 phosphorylated on CTD
4. Spt4/5 (DSIF) and NELF associated with paused polymerase ( ChIP; + required
to recapitulate pause in vitro )
What triggers release to productive elongation?
1)pTEFb phosphorylates Spt5 releasing it to move
with RNA P; and a subunit of NELF, causing dissociation
Paused
polymerase
2) Backtracking relieved ( SII)
Genome wide studies sequencing 5’ capped short mRNAs found them associated with ~30%
of all genes in Drosophila; positions of their 3’ ends correspond to positions of stalled
polymerase, and were also regions of high GC content; length of short mRNAs increases
when SII is depleted suggesting that paused polymerases had backtracked and their mRNAs
had been cleaved by SII
Adelman, Science, Cell 2010
Potential roles of Paused Polymerase
NRG ( 2012) 13: 720
Spt4/5 is also connected to other elongation complexes
Using activity based assay, Spt4/5, PAF and Tat-SF1 required for efficient elongation (DNA template)
PAF
Spt4/5
Phosphorylation of Spt5 CTD by Bur-1 required for PAF
entry into elongation complex
Tat-SF1
Physical interaction
PAF
Using chromatin template and completely
reconstituted factors, PAF stimulates elongation
synergistically with TFIIS (independent of other
activities of the PAF complex)
PAF
∆PAF ∆TFIIS
TFIIS
Physical interaction
Synthetic lethal
Each elongation factor also interacts with RNAP
NusG may also mediate ribosome/ RNAP interaction
50 µM
CTD
NusE
NusE is ribosomal protein S10, and structural studies indicate that its
binding site would be exposed when S10 is part of the ribosome. This
protein protein interaction could connect these two major macromolecular
machines
Altering translation rate alters the transcription rate
Condition
+ chloramphenicol ( 1µg/ml)
Slow ribosome (streptomycin dependent)
Slow ribosome (+ streptomycin)
translation rate
14 aa/sec
9 aa/sec
6 aa/sec
10 aa/sec
transcription rate
42 nt/sec
27 nt/sec
19 nt/sec
31 nt/sec
Footprinting studies show that the presence of a ribosome behind
RNA polymerase prevents backtracking!
This could be a general mechanism to couple the rates of transcription and translation