Biochemistry 201Biological Regulatory Mechanisms

Transcription and Its Regulation

January 22 –Mechanism of Transcription InitiationJanuary 24– Mechanism of Transcription ElongationJanuary 28– Control of Transcription in BacteriaJanuary 31– Control of Transcription in Eukaryotes

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. 377-4142.2. 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

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 Subunit. Cell 147: 1257 – 1269Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription: –DNA Interaction. Cell: 147: 1218-1219

***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.

The accompanying minireview is helpfulNickels, B.E. and Hochschild, A. (2004) Regulation of RNA Polymerase through the Secondary Channel. Cell 118:281-284

Key Points1. Multisubunit RNA polymerases are conserved among all organisms

2. RNA polymerases cannot initiate transcription on their own. In bacteria 70 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 + 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, 70 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

Transcription/Splicing/Translation ProvideA Large Range of Protein Concentrations

I. RNA polymerases

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 R

NA

P

Structure of RNAP in the three domains 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 conserved

Archaeal/eukaryotic

Bacteria Archaea Eukarya

Transcription

Evolutionary relationships of general transcription factorsEvolutionary relationships of general transcription factors

Initiation

GreTranscript cleavage

Elongation

LUCA 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

The Initiating Form of RNA Polymerase

‘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 column

salt

OD

28

0

1

2

Act

ivit

y (

*ATP)

CT D

NA

Fraction #

SDS gel analysis Peak 1 Peak 2

'

increases rate of initiation

g

Tra

nsc

ripti

on

D

NA Assay:

incorporationP

ATP

(2) Bacterial promoters

There are several flavors of promoters

and recruit RNAP to promoter DNA

(3) undergoes a large conformational change upon binding to RNA polymerase

Free doesn’t bind DNA in holoenzyme positioned for DNA recognition Sorenson; 2006

is positioned for DNA recognition

is positioned to affect key activities of RNA polymerase

Surprising structural similarity between the initiating

forms of bacterial and eukaryotic RNAP

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

D Kostrewa et al. Nature 462, 323-330 (2009) doi:10.1038/nature08548

TFIIB has a central role in initiation similar to that of

Recruits Pol II to promoter: N-terminus binds Pol II; C terminus binds TBP and DNA

TFIIB structure

Role in promoter opening; B linker mutants recruit PolII but cant strand open or initiate

Role in selection of TSS ( Inr): B reader mutants

Blocks elongating RNA chain: B reader

Crystal structure of TFB + RNA polymerase--archae

Topological similarities in /TFIIB binding to RNAP

B reader ( 3.2): both in exit channel and near active site; start site selection

B ribbon (4):both bind flap tip helix

B linker (2): both bind coiled -coil and rudder; both involved in strand opening

B core (3)

D Kostrewa et al. Nature 462, 323-330 (2009) doi:10.1038/nature08548

TFIIB and bound to RNA polymerase show surprising similarity. Analogously placed regions have similar functions

Initiating RNAP must open DNA to permit transcription:Formation of the open complex

Steps in transcription initiation

KB Kf

initial binding

“isomerization”

Abortive Initiation

ElongatingComplex RPoRPcR+P

NTPs

A detailed look at a prokaryotic promoter

Sequence Logos-35 logo -10 logo

TT TT AAGG CC AA TT AA TT AA AA TT15-

19nucleotides

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

Recognition of the prokaryotic promoter

Approach

1. Determine a high resolution structure of 2 bound to non-template strand of the -10 element

2. Determine whether this structure represents the “initial binding state” or endpoint state

Schematic

Promoter escape

is positioned to affect key activities of RNA polymerase

Promoter escape and Abortive Initiation

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 promoter contact?

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

Initial transcription involves 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

Validation of the prediction that occlusion of the RNA exit channel promotes “abortive initiation”

#1: transcription by holoenzyme with full-length #2: transcription by holoenzyme with truncated at Region 3.2: lacks in the RNA exit channel

Murakami, Darst 2002

is positioned to affect key activities of RNA polymerase