transcription cycle anti- 's microcin j25 rifampicin grea greb activators trcf (mfd)
TRANSCRIPT
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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)
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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)
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Miller spread
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RNAP
!TRCFTRCF
Uvr system(NER)TRCF
Transcription Repair Coupling Factor (TRCF, mfd)
Selby & Sancar (1993) Science 260, 53.
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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.
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Rescue• Transcript cleavage (stimulated by GreA/GreB)• Slide forward
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Park et al. (2002) Cell 109, 757
Requirements for TRCF activity on the elongation complex
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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
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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)
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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.
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Deaconescu et al. (2006) Cell 124, 507.
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Deaconescu et al. (2006) Cell 124, 507.
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R/Rfree = 0.25/0.29 (3.2 Å resolution)APS NE-CAT-8BM
APS SBC-19IDDeaconescu et al. (2006)
Cell 124, 507.
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QuickTime™ and aCompact Video decompressorare needed to see this picture.
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Singleton et al. (2001) Cell 107, 79Deaconescu et al. (2006) Cell 124, 507.
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UvrB homology module, UvrA recruitment, and the role of D7
Truglio et al. (2002) EMBO
J. 23, 2498
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• necessary for RNAP binding (Selby & Sancar, 1995)• binds RNAP β subunit 19-142 (Park et al., 2002)
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DNA-binding and ATPase activity of TRCF-L499R
mutant intact
Deaconescu et al. (2006) Cell 124, 507.
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Deaconescu et al. (2006) Cell 124, 507.
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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
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Dürr et al. (2005) Cell 121, 363
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• 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?
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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)
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C. Mfd
B. Rho
A. Intrinsicterminator
Figure 1Bacterial transcription termination
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Roadblock repression assay
(Chambers et al., 2003, Nucleic Acids Res. 31, 6409)
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DNA-binding activity of TRCF-L499R mutant intact
(also ATPase activity)
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Epshtein & Nudler (2003) Cooperation between RNA polymerase molecules in transcription elongation, Science 300, 801.