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Transcription and Its Regulation
January 21 –Mechanism of Transcription InitiationJanuary 23– Regulation of of Transcription InitiationJanuary 27–Mechanism and regulation of Transcription ElongationJanuary 30– In class discussion of problem set
Mechanism of Transcription Initiation
ReferencesI. General
Chapter 12 of Molecular Biology of the Gene 6 th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M, Losick, R. 377-414.
2. ReviewsMurakami KS, Darst SA. (2003) Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 13:31-9.
Campbell, E, Westblade, L, Darst, S., (2008) Regulation of bacterial RNA polymerase factor activity: a structural perspective. Current Opinion in Micro. 11:121-127
Herbert, KM, Greenleaf, WJ, Block, S. (2008) Single-Molecule studies of RNA polymerase: Motoring Along. Annu Rev Biochem. 77:149-76.
Werner, Finn and Dina Grohmann (201). Evolution of multisubunit RNA polymerases in the three domains of life. Nature Rev. Microbiology 9: 85-98
Grunberg, S. and Steven Hahn (2013) Structural Insights into transcription initiation by RNA polymerase II. TIBS 38: 603-11.
3. Studies of Transcription InitiationRoy S, Lim HM, Liu M, Adhya S. (2004) Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step. EMBO J. 23:869-75.
Sorenson MK, Darst SA. (2006).Disulfide cross-linking indicates that FlgM-bound and free sigma28 adopt similar conformations. Proc Natl Acad Sci U S A. 103:16722-7.
Young BA, Gruber TM, Gross CA. (2004) Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting. Science. 303:1382-1384
*Kapanidis, AN, Margeat, E, Ho, SO,.Ebright, RH. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science. 314:1144-1147.
Revyakin A, Liu C, Ebright RH, Strick TR (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science. 314: 1139-43.
Murakami KS, Masuda S, Campbell EA, Muzzin O, Darst SA (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science. 296:1285-90.
Kostrewa D, Zeller ME, Armache KJ, Seizl M, Leike K, Thomm M, Cramer P.(2009) RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature. 462:323-30.
Discussion Paper**Feklistov A and Darst, SA (2011) Structural basis for Promoter -10 Element recognition by the Bacterial RNA Polymerase s Subunit. Cell 147: 1257 – 1269Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription:s –DNA Interaction. Cell: 147: 1218-1219
Reviews
Articles:Chromosome conformation capture (CCC) technologies
de Wit, E. and de Laat, W. (2012) A decade of 3C technologies: insights into nuclear organization. Genes Dev. 26: 11-24.
ElongationBBA2013-- Issue 1874 devoted to reviews of transcription elongation
General Transcription Factors
Matsui, T., Segall, J., Weil, P.A., and Roeder, R.G. (1980) Multiple factors required for accurate initiation of transcription by purified RNA polymerase II. J Biol Chem 255: 11992-11996.
Thomas, M.C., & Chiang, C.M. (2006). The general transcription machinery and general cofactors. Critical reviews in Biochemistry & Molecular Biology, 41(3), 105-78.
Muller, F, Demeny, MA, & Tora, L. (2007). New problems in RNA polymerase II transcription initiation: matching the diversity of core promoters with a variety of promoter recognition factors. The Journal of Biological Chemistry, 282(20), 14685-9.
Mediator and Other Components
*Kornberg, R.D. (2005) Mediator and the mechanism of transcriptional activation. Trends in Biochemical Sciences 30:235-239.
Fan, X, Chou, DM, & Struhl, K. (2006). Activator-specific recruitment of Mediator in vivo. Nature Structural & Molecular Biology, 13(2), 117-20.
Sikorski TW and Buratowski. (2009). The basal initiation machinery: Beyond the general transcription factors. Current Opinion in Cell Biology. 21 344-351.
Key Points1. Multisubunit RNA polymerases are conserved among all organisms
2. RNA polymerases cannot initiate transcription on their own. In bacteria s70 is required to initiate
transcription at most promoters. Among other functions, it recognizes the key features of most bacterial promoters, the -10 and -35 sequences.
2. E. coli RNA polymerase holoenzyme, (core + s) finds promoter sequences by sliding along DNA and by transfer from one DNA segment to another. This behavior greatly speeds up the search for specific DNA sequences in the cell and probably applies to all sequence-specific DNA-binding proteins.
3. Transcription initiation proceeds through a series of structural changes in RNA polymerase, s70 and DNA.
4. A key intermediate in E. coli transcription initiation is the open complex, in which the RNA polymerase holoenzyme is bound at the promoter and ~12 bp of DNA are unwound at the transcription startpoint. Open complex formation does not require nucleoside triphosphates. Its presence can be monitored by a variety of biochemical and structural techniques.
5. Recognition of the -10 element of the promoter DNA is coupled with strand separation
6. When the open complex is given NTPs, it begins the ‘abortive initiation’ phase, in which RNA chains of 5-10 nucleotides are continually synthesized and released.
7. Through a “DNA scrunching” mechanism the energy captured during synthesis of one of these short transcripts eventually breaks the enzyme loose from its tight connection to the promoter DNA, and it begins the elongation phase.
7. Aspects of the mechanism of initiation are likely to be conserved in eukaryotic RNA polymerase
rRNAs snRNAs miRNAs
Other non-coding RNAs (e.g. telomerase RNA)
mRNAs
translation
proteins
transcription
(RNA processing)
Transcription is Important
Cellular RNA polymerases in all living organisms are evolutionary related
A common structural and functional frame work of transcription in the three domains of life
LUCA-Last universal common ancestor
Sub
units
of
RN
AP
Structure of RNAP in the three domains
Werner and Grohmann (2011),Nature Rev Micro 9:85-98
Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities
Universally conservedArchaeal/eukaryotic
Bacteria Archaea Eukarya
Transcription
Eukaryotic Cells have three RNA polymerases
TYPE OF POLYMERASE GENES TRANSCRIBED
RNA polymerase I 5.85, 18S, and 28S rRNA genes
RNA polymerase II all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, and some snRNA genes
RNA polymerase III tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs
The rRNAs are named according to their “S” values, which refer to their rate of sedimentation in an ultra-centrifuge. The larger the S value, the larger the rRNA.
Evolutionary relationships of general transcription factors
s
Initiation s
GreTranscript cleavage
ElongationLUCA may have had elongating, not initiating RNA polymerase
II. Challenges in initiating transcription
1. RNAP is specialized to ELONGATE, not INITIATE
2. Initiating RNAP must open DNA to permit transcription
3. RNAP must leave promoter—abortive initiation
‘holoenzyme’
'
KD ~ 10-9 M+
‘core’}
Can begin transcription on
promoters and can elongate
}Can elongate but
cannot begin transcription at
promoters
factor is required for bacterial RNA polymerase to initiate transcription on promoters
'
(1) The discovery of initiation factors
How was discovered (Burgess, 1969)A. Assay for RNA polymerase:
E.coli lysate
buffer
*ATPCTPGTPUTP
Calf thymus DNA
Look for incorporation of *ATP into RNA chains
B. Initial purification Lysate
various fractionation steps (DEAE column, glycerol gradient etc)
Active fractions identified by assay
Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had no activity on DNA
Peak 1 restored activity
C. Improved purification of RNA polymerase:
Improved fractionationlysate
phosphocellulose columnsalt
OD 2
80
1
2
Activ
ity (*
ATP)
CT D
NA
Fraction #
SDS gel analysis Peak 1 Peak 2
'
increases rate of initiation
g
Tran
scrip
tion
DNA Assay:
incorporation P ATP
(3) s undergoes a large conformational change upon binding to RNA polymerase
Free doesn’t bind DNA in holoenzyme positioned for DNA recognition Sorenson; 2006
Is the -10 promoter element recognized as Duplex or SS DNA?
-10 logo-35 logo
Helix-turn-helix in Domain 4Recognizes -35 as duplex DNA
The Strand Separation/Melting Step
Approach
1. Determine a high resolution structure of s2 bound to non-template strand of the -10 element
2. Determine whether this structure represents the “initial binding state” or endpoint state
Schematic
Transcription Initiation by PolII requires many General Transcription Factors
RNA Pol II+ NTPs+ DNA containing a real promoter
NO TRANSCRIPTION
promoter
RNA Pol II+ NTPs+ DNA with real promoter
TRANSCRIPTION INITIATION and ELONGATION
nuclear extract
Purification scheme for partially purified general transcription factors. Fractionation of HeLa nuclear extract (Panel A) and nuclear pellet (Panel B) by column chromatography and the molar concentrations of KCl used for elutions are indicated in the flow chart, except for the Phenyl Superose column where the molar concentrations of ammonium sulfate are shown. A thick horizontal (Panel A) or vertical (Panel B) line indicates that step elutions are used for protein fractionation, whereas a slant line represents a linear gradient used for fractionation. The purification scheme for pol II, starting from sonication of the nuclear pellet, followed by ammonium sulfate (AS) precipitation is shown in Panel B. (Figures are adapted from Flores et al., 1992 and from Ge et al., 1996)
NAME # OF SUBUNITS FUNCTION
TFIIA 3 Antirepressor; stabilizes TBP-TATA complex; coactivator
TFIIB 1 Recognizes BRE;Start site selection; stabilize TBP-TATA; pol II/TFIIF recruitment
TFIID TBP 1 Binds TATA box; higher eukaryotes have multiple TBPs TAFs ~10 Recognizes additional DNA sequences; Regulates TBP binding; Coactivator;
Ubiquitin-activating/conjugating activity; Histone acetyltransferase; multiple TAFs
TFIIF 2 Binds pol II; facilitates pol II promoter recruitment and escape; Recruits TFIIE and TFIIH; enhances efficiency of pol II elongation
TFIIE 2 Recruits TFIIH; Facilitates forming initiation-competent pol II; promoter clearance TFIIH 9 ATPase/kinase activity. Helicase: unwinds DNA at transcription startsite; kinase
phosphorylates ser5 of RNA polymerase CTD; helps release RNAP from promoter
Transcription Initiation by RNA Pol II
The stepwise assembly of the Pol II preinitiation complex is shown here. Once assembled at the promoter, Pol II leaves the preinitiation complex upon addition of the nucleotide precursors required for RNA synthesis and after phosphorylation of serine resides within the enzyme’s “tail”.
PIC = preinitiation complex
The first two steps of Eukaryotic transcription
Many archae have a proliferation of TBPs and TFBs, suggesting that they provide choice in promoters, akin to alternative s.
In archae, TBP and TFB are sufficient for formation of the pre-initiation complex (PIC), suggesting that they are key to the mechanism of transcription initiation in eukaryotes
Promoter
TFBTBP
The Pol II promoter has many recognition regions
Positions of various DNA elements relative to the transcription start site (indicated by the arrow above the DNA). These elements are:
BRE (TFIIB recognition element); there is also a second BRE site downstream of TATA
TATA (TATA Box);
Inr (initiator element);
DPE (downstream promoter element);
DCE (downstream core element).
MTE (motif ten element; not shown) is located just upstream of the DPE.
Steps in transcription initiation
KB Kf
initial binding
“isomerization”
Abortive Initiation
ElongatingComplex RPoRPcR+P
NTPs
KB Kf
initial binding
“isomerization”
Abortive Initiation
ElongatingComplex RPoRPcR+P
NTPs
Abortive Initiation and Promoter escape
During abortive initiation, RNAP synthesizes many short transcripts, but reinitiates rapidly.
How can the active site of RNAP move forward along the DNA while maintaining
contact with the promoter?
Förster (fluorescence) resonance energy transfer (FRET) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution
Experimental set-up for single molecule FRET: Single transcription complexes labeled with a fluorescent donor (D, green) and a fluorescent acceptor (A, red) are illuminated as they diffuse through a femtoliter-scale observation volume (green oval; transit time ~1 ms); observed in confocal microscope
Using single molecule FRET to monitor movement of RNAP and DNA
Three models for Abortive initiation
#1
Predicts expansion and contraction of RNAP
Predicts expansion and contraction of DNA
Predicts movement of both the RNAP leading and trailing edge relative to DNA
#2
#3
A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)
Initial transcription involves DNA scrunching
Lower E* peak is free DNA; higher E* peak is DNA in open complex; distance is shorter because RNAP
induces DNA bending
Open complex
Initial transcription involves DNA scrunching
Higher E* in Abortive initiation complex than open complex results from DNA scrunching
Open complex
Abortive initiation complex
At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)].
The energy accumulated in the DNA scrunched “stressed intermediate could disrupt interactions between RNAP,
and the promoter, thereby driving the transition from initiation to elongation