Location of close contacts between Escherichia coli RNA polymerase and guanine residues at promoters either with or without consensus -35 region sequences

Methylation-interference assays have been used to identify guanine residues that make important contacts with RNA polymerase during open-complex formation at two related Escherichia coli promoters. Methylation of lower-strand G-31 at a gal consensus promoter completely prevents complex formation, while modification of upper-strand G-33 has no detectable effect. At galP1, which lacks a consensus -35 region, modification of lower-strand G-33 and upper-strand G-14 reduces, but does not prevent, complex formation. G-33 is the only guanine residue in the -35 region of galP1 where modification interferes with open-complex formation. Since this guanine residue is not protected in open complexes, we conclude that its modification causes alteration of, or interference with, a transient contact during the transcription initiation pathway.

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The organization of open complexes between Escherichia coli RNA polymerase and DNA fragments carrying promoters either with or without consensus −35 region sequences

Biochemical Journal ◽

10.1042/bj2700141 ◽

1990 ◽

Vol 270 (1) ◽

pp. 141-148 ◽

Cited By ~ 25

Author(s):

B Chan ◽

A Spassky ◽

S Busby

Keyword(s):

Escherichia Coli ◽

Complex Formation ◽

Rna Polymerase ◽

Transcription Initiation ◽

Point Mutations ◽

Dna Fragments ◽

Transcription Start ◽

Footprint Analysis ◽

Open Complex Formation ◽

Promoter Dna

Transcription initiation at the Escherichia coli galP1 promoter does not depend on specific nucleotide sequences in the -35 region. Footprint analysis of transcriptionally competent complexes between E. coli RNA polymerase and DNA fragments carrying galP1 shows that RNA polymerase protects sequences as far upstream as -55, whereas sequences around the -35 region are exposed. In contrast, with galP1 derivatives carrying -35 region sequences resembling the consensus, RNA polymerase protects bases as far as -45, and the -35 region is fully protected. Taken together, our data suggest that the overall architecture of RNA polymerase-promoter complexes can vary according to whether or not consensus -35 region sequences are present; in the absence of these sequences, open complex formation requires distortion of the promoter DNA. However, the unwinding of promoter DNA around the transcription start is not affected by the nature of the -35 region sequence. With a galP1 derivative carrying point mutations in the spacer region that greatly reduce promoter activity, the protection of bases by RNA polymerase around the -10 sequence and transcription start site is reduced. In contrast, protection of the region upstream of -25 is unaffected by the spacer mutations, although sequences from -46 to -54 become hypersensitive to attack by potassium permanganate, indicating severe distortion or kinking of this zone. We suggest that, with this galP1 derivative, RNA polymerase is blocked in a complex that is an intermediate on the path to open complex formation.

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Open complex formation during transcription initiation at the Escherichia coli galP1 promoter: The role of the RNA polymerase subunit at promoters lacking an UP-element

Nucleic Acids Research ◽

10.1093/nar/27.9.2051 ◽

1999 ◽

Vol 27 (9) ◽

pp. 2051-2056 ◽

Cited By ~ 12

Author(s):

H. D. Burns ◽

S. D. Minchin ◽

A. Ishihama

Keyword(s):

Escherichia Coli ◽

Complex Formation ◽

Rna Polymerase ◽

Transcription Initiation ◽

Open Complex Formation

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CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase

Nucleic Acids Research ◽

10.1093/nar/gkz449 ◽

2019 ◽

Vol 47 (13) ◽

pp. 6685-6698 ◽

Cited By ~ 10

Author(s):

Drake Jensen ◽

Ana Ruiz Manzano ◽

Jayan Rammohan ◽

Christina L Stallings ◽

Eric A Galburt

Keyword(s):

Mycobacterium Tuberculosis ◽

Complex Formation ◽

Rna Polymerase ◽

Transcription Initiation ◽

Promoter Escape ◽

Rate Limiting Step ◽

Nucleotide Incorporation ◽

Transcriptional Regulatory ◽

Open Complex Formation ◽

Transcriptional Regulatory Mechanisms

Abstract The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics.

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