Investigations of the modular structure of bacterial promoters

Bacterial RNA polymerase holoenzyme carries different determinants that contact different promoter DNA sequence elements. These contacts are essential for the recognition of promoters prior to transcript initiation. Here, we have investigated how active promoters can be built from different combinations of elements. Our results show that the contribution of different contacts to promoter activity is critically dependent on the overall promoter context, and that certain combinations of contacts can hinder transcription initiation.

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Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences

10.20944/preprints202008.0548.v1 ◽

2020 ◽

Author(s):

Masahiko Imashimizu ◽

Yuji Tokunaga ◽

Ariel Afek ◽

Hiroki Takahashi ◽

Nobuo Shimamoto ◽

...

Keyword(s):

Rna Polymerase ◽

Dna Sequences ◽

Repetitive Sequence ◽

Transcription Initiation ◽

High Throughput Sequencing ◽

Thermal Fluctuations ◽

A Genome ◽

Sequence Elements ◽

Promoter Dna

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences enriched with thymine bases represent the signal inducing abortive transcription. On the other hand, certain repetitive sequence elements broadly embedded in promoter regions constitute the signal inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we also suggest that repetitive sequence elements of promoter DNA modulate the rigidity of its double-stranded form, which profoundly influences the reaction coordinates of the productive initiation via pausing.

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Mutant Forms of Salmonella typhimuriumς54 Defective in Transcription Initiation but Not Promoter Binding Activity

Journal of Bacteriology ◽

10.1128/jb.181.11.3351-3357.1999 ◽

1999 ◽

Vol 181 (11) ◽

pp. 3351-3357 ◽

Cited By ~ 10

Author(s):

Mary T. Kelly ◽

Timothy R. Hoover

Keyword(s):

Complex Formation ◽

Rna Polymerase ◽

Transcription Initiation ◽

Atp Hydrolysis ◽

Random Mutagenesis ◽

Binding Activity ◽

Mutant Proteins ◽

Promoter Dna ◽

Mutant Forms ◽

Rna Polymerase Holoenzyme

ABSTRACT Transcription initiation with ς54-RNA polymerase holoenzyme (ς54-holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. ς54’s binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation. Following random mutagenesis, we isolated eight mutant forms of Salmonella typhimurium ς54 that were deficient in transcription initiation but still directed ς54-holoenzyme to the promoter to form a closed complex. Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for ς54-holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of ς54 at some point after closed-complex formation. These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.

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Conformational properties of Bacillus subtilis RNA polymerase σA factor during transcription initiation

Biochemical Journal ◽

10.1042/bj2940043 ◽

1993 ◽

Vol 294 (1) ◽

pp. 43-47 ◽

Cited By ~ 3

Author(s):

B Y Chang ◽

R H Doi

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Trypsin Digestion ◽

Tryptic Fragment ◽

The Core ◽

Core Enzyme ◽

Conformational Properties ◽

Promoter Dna ◽

Rna Polymerase Holoenzyme ◽

Dna Complex

By the use of a partial proteolysis method and Western-blot analysis, the conformational properties of Bacillus subtilis sigma A factor in the transcription initiation stage were studied. From a comparison of the trypsin-digestion patterns of free sigma A and of sigma A associated with core enzyme, it was found that the production of 45 kDa sigma A tryptic-derived fragment was enhanced when sigma A was associated with the core enzyme. More importantly, a 40 kDa sigma A tryptic-derived fragment was found exclusively in this associated state. Based on the change of the digestion kinetics when producing the 45 kDa tryptic fragment and the generation of this new 40 kDa tryptic fragment from sigma A, it was apparent that a conformation change of sigma A occurred during the association of sigma A with the core enzyme. Also, similar patterns were found for the sigma A present in the holoenzyme-promoter DNA complex. These findings suggest that no further distinctive conformational change of sigma A occurs at the step of RNA polymerase holoenzyme and promoter DNA complex formation. Trypsin-digestion patterns of sigma A in different RNA polymerase holoenzyme and promoter DNA complexes were also studied. The presence of similar trypsin digestion-patterns of sigma A in those complexes strongly supports the idea that a similar sigma A conformation is used in the recognition of different sigma A-type promoters and the formation of different open complexes.

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Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme

Journal of Biological Chemistry ◽

10.1074/jbc.m114.584037 ◽

2014 ◽

Vol 289 (35) ◽

pp. 24549-24559 ◽

Cited By ~ 106

Author(s):

Ritwika S. Basu ◽

Brittany A. Warner ◽

Vadim Molodtsov ◽

Danil Pupov ◽

Daria Esyunina ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Structural Basis ◽

Bacterial Rna ◽

Rna Polymerase Holoenzyme

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Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences

Biomolecules ◽

10.3390/biom10091299 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1299 ◽

Cited By ~ 2

Author(s):

Masahiko Imashimizu ◽

Yuji Tokunaga ◽

Ariel Afek ◽

Hiroki Takahashi ◽

Nobuo Shimamoto ◽

...

Keyword(s):

Rna Polymerase ◽

Dna Sequences ◽

Repetitive Sequence ◽

Transcription Initiation ◽

High Throughput Sequencing ◽

Thermal Fluctuations ◽

A Genome ◽

Sequence Elements ◽

Promoter Dna

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on the sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences, when enriched with thymine bases, contain the signal for inducing abortive transcription, whereas certain repetitive sequence elements embedded in promoter regions constitute the signal for inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we suggest that repetitive sequence elements within the promoter DNA modulate the nonlocal base pair stability of its double-stranded form. This stability profoundly influences the reaction coordinates of the productive initiation via pausing.

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