Transcription cycle Anti- 's Microcin j25 Rifampicin GreA GreB Activators TRCF (mfd)

Transcription cycle

σ

α2ββ'(core)

α2ββ'σ

(holoenzyme)

α2ββ'σα2ββ'σ

NTPs

α2ββ'σ

α2ββ'σ

α2ββ'

α2ββ'

-35 -10

-35-10

-35-10

-35 -10

Terminator

+1

Promoter Location

closedpromotercomplex

Formation of Open Promoter Complex

AbortiveInitiation

PromoterClearance

Elongation

Termination

Anti-σ's

Microcin j25

Rifampicin

GreAGreB

Activators

TRCF(mfd)

Transcription cycle

σ

α2ββ'(core)

α2ββ'σ

(holoenzyme)

α2ββ'σα2ββ'σ

NTPs

α2ββ'σ

α2ββ'σ

α2ββ'

α2ββ'

-35 -10

-35-10

-35-10

-35 -10

Terminator

+1

Promoter Location

closedpromotercomplex

Formation of Open Promoter Complex

AbortiveInitiation

PromoterClearance

Elongation

Termination

TRCF(mfd)

Miller spread

RNAP

!TRCFTRCF

Uvr system(NER)TRCF

Transcription Repair Coupling Factor (TRCF, mfd)

Selby & Sancar (1993) Science 260, 53.

Selby & Sancar (1993) Science 260, 53.Selby & Sancar (1995) J. Biol. Chem. 270, 4882.

Mahdi et al. (2003) EMBO J. 22, 724.Chambers et al. (2003) Nucleic Acids Res. 31, 6409.

Rescue• Transcript cleavage (stimulated by GreA/GreB)• Slide forward

Park et al. (2002) Cell 109, 757

Requirements for TRCF activity on the elongation complex

RNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAP

RNAP

translocation domains

wedge domain

RNAP binding domain

RecG

Singleton et al. (2001) Cell 107, 79

TRCF

Park et al. (2002) Cell 109, 757

Alexandra Deaconescu

Collaborators

Anna L. Chambers, Abigail J. Smith, Nigel J. Savery (University of Bristol, Bristol, UK)

Bryce E. Nickels, Ann Hochschild (Harvard Medical School)

Data collection and model refinement statistics for apo Mfd

Space group: C2; 2 molecules per asymmetric unit SeMet1 SeMet2Cell dimensions (Å): a=151.7 b=162.2 c=162.1 a=151.9 b=162.0 c= 161.7 β= 104.9° β= 105.1°Wavelengt (h Å) 0.98166 0.97899Resolut ion( ) 40-4.0 40Å -3.2Reflecti onsmeasure 228,334 300,181d(unique) 62,430 122,388Completene ss(%) 99.6 99.9(i n oute r shell) 100.0 99.9Mea n I/σ(I) 10.9 12.4(i n oute r shell) 2.9 2.5Rs ym

a(%) 12.9 0.08(i n oute r shell) 50.2 37.7

S AD Figu re o f Meri 0.17tRefinement dat ase : t SeM 2 (|ET F| >0)R-facto r 23.4% R-fre efact 29.5%orR. .ms. d bondle ngth( )Å : 0.011R. .ms. d bondangle s (°): 1.49

aRsym=Σ|I-<I>|/Σ , I wher e =I obse rvedintensit , <y I>=avera geintensi ty o f multipleobservati ons o f symmetr -y relat ed reflectionsbRcryst=Σ|Fo - FcI/Σ|Fo|, whe reFo andFc ar e obser ved andcalculate dstructur efactors,respectively

Experimental electrondensity map (after solvent modification)

Deaconescu et al. (2006) Cell 124, 507.

Deaconescu et al. (2006) Cell 124, 507.

Deaconescu et al. (2006) Cell 124, 507.

R/Rfree = 0.25/0.29 (3.2 Å resolution)APS NE-CAT-8BM

APS SBC-19IDDeaconescu et al. (2006)

Cell 124, 507.

QuickTime™ and aCompact Video decompressorare needed to see this picture.

Singleton et al. (2001) Cell 107, 79Deaconescu et al. (2006) Cell 124, 507.

UvrB homology module, UvrA recruitment, and the role of D7

Truglio et al. (2002) EMBO

J. 23, 2498

• necessary for RNAP binding (Selby & Sancar, 1995)• binds RNAP β subunit 19-142 (Park et al., 2002)

DNA-binding and ATPase activity of TRCF-L499R

mutant intact

Deaconescu et al. (2006) Cell 124, 507.

Deaconescu et al. (2006) Cell 124, 507.

Park et al. (2002) Cell 109, 757

A. L. Chambers, A.J. Smith, N. J. Savery

B. Nickels, A. HochschildLeu499

Park et al. (2002) Cell 109, 757Smith & Savery (2005) Nucleic Acids Res. 33, 755

Dürr et al. (2005) Cell 121, 363

• TRCF - compact arrangement of structural domains linked by long, flexible linkers - appears ‘primed’ for large scale conformational changes (translocation module, UvrA binding surface).

? Nature and timing of TRCF conformational changes during the steps of TRCF-mediated transcription-coupled repair reaction? (recognition of a stalled RNAP; forward translocation of the RNAP to the transcription block; RNAP release and transcript termination; recruitment of the NER machinery to the site)

? Details of protein/protein interactions involving TRCF? (TRCF/RNAP and TRCF/NER machinery)

? Termination mechanism?

Darst LabMatthew BickElizabeth CampbellLinda (Madge) Champagnie

Alexandra DeaconescuRaji EdayathumangalamDeepti JainValerie LamourWilliam LaneFred LeonNatacha OpalkaGeorgia PatikoglouSheng WangLars WestbladeKelly-Anne Wilson

Anna L. Chambers, Abigail J. Smith, Nigel J. Savery (University of Bristol)

Bryce E. Nickels, Ann Hochschild (Harvard Medical School)

Chris Roberts, Jeff Roberts (Cornell University)

Chris Selby, Aziz Sancar (University of North Carolina, Chapel Hill)

N. Sukumar , M. Capel, S. Ealick (APS NE-CAT-8BM)

A. Joachimiak, S. Grinnell (APS SBC-19ID)

C. Mfd

B. Rho

A. Intrinsicterminator

Figure 1Bacterial transcription termination

Roadblock repression assay

(Chambers et al., 2003, Nucleic Acids Res. 31, 6409)

DNA-binding activity of TRCF-L499R mutant intact

(also ATPase activity)

Epshtein & Nudler (2003) Cooperation between RNA polymerase molecules in transcription elongation, Science 300, 801.