Passage of RNA polymerase from open complex to elongation mode at the Escherichia coli lacUV5 promoter: nucleolytic hypersensitivity as a probe for complex conformational changes

Biochemistry ◽  
1992 ◽  
Vol 31 (43) ◽  
pp. 10502-10509 ◽  
Author(s):  
Annick Spassky
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Lacuv5 Promoter

scholarly journals Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

2014 ◽  
Vol 112 (3) ◽  
pp. 743-748 ◽  
Author(s):  
Yara X. Mejia ◽  
Evgeny Nudler ◽  
Carlos Bustamante
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Conformational Changes ◽  
Elongation Rate ◽  
Nucleoside Triphosphate ◽  
Transcription Rate ◽  
Nucleotide Analogs ◽  
Catalytic Center ◽  
Trigger Loop

Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP’s pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity.

Conformational Changes of Escherichia coli σ54-RNA-Polymerase upon Closed–Promoter Complex Formation

2005 ◽  
Vol 354 (2) ◽  
pp. 201-205 ◽  
Author(s):  
Pampa Ray ◽  
Richard J. Hall ◽  
Robert D. Finn ◽  
Shaoxia Chen ◽  
Ardan Patwardhan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Complex Formation ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Promoter Complex

Interactions of transcription inhibitors with the Escherichia coli RNA polymerase-lacUV5 promoter open complex

Biochemistry ◽  
1994 ◽  
Vol 33 (8) ◽  
pp. 2262-2268 ◽  
Author(s):  
Abhijit Mazumder ◽  
David M. Perrin ◽  
David McMillin ◽  
David S. Sigman
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Inhibitors ◽  
Lacuv5 Promoter

scholarly journals Using Disulfide Bond Engineering To Study Conformational Changes in the β′260-309 Coiled-Coil Region of Escherichia coli RNA Polymerase during σ70 Binding

2002 ◽  
Vol 184 (10) ◽  
pp. 2634-2641 ◽  
Author(s):  
Larry C. Anthony ◽  
Alan A. Dombkowski ◽  
Richard R. Burgess
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Disulfide Bond ◽  
Conformational Changes ◽  
Coiled Coil ◽  
Mrna Synthesis ◽  
Promoter Sequences ◽  
Coiled Coil Region

ABSTRACT RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the β′ subunit (β′260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with σ70 both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to σ70. In this work, we engineered a disulfide bond within β′240-309 that locks the β′ coiled-coil region in the coiled-coil conformation, and we show that this “locked” peptide is able to bind to σ70. We also show that the locked coiled-coil is capable of inducing a conformational change within σ70 that allows recognition of the −10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding σ70 or upon recognition of the −10 nontemplate strand of DNA.

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