scholarly journals Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

2021 ◽  
Author(s):  
Subhas C Bera ◽  
Pim P. B. America ◽  
Santeri Maatsola ◽  
Mona Seifert ◽  
Eugeniu Ostrofet ◽  
...  
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Magnetic Tweezers ◽  
Energy Scale ◽  
Initiation Factor ◽  
Dissociation Pathway ◽  
Domains Of Life ◽  
Open Complex Formation

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor σ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high – throughput single-molecule magnetic tweezers. We probed the key protein – DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RPO is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence – independent interactions between the DNA and the holoenzyme, driven by a non – Coulombic effect consistent with the non-template DNA strand interacting with σ and the RNAP β subunit. The temperature dependence provides the energy scale of open complex formation and further supports the existence of additional intermediates.

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

2019 ◽  
Vol 47 (13) ◽  
pp. 6685-6698 ◽  
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.

scholarly journals Structural basis of RNA polymerase inhibition by viral and host factors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Simona Pilotto ◽  
Thomas Fouqueau ◽  
Natalya Lukoyanova ◽  
Carol Sheppard ◽  
Soizick Lucas-Staat ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Environmental Changes ◽  
Initiation Factor ◽  
Structural Basis ◽  
Regulation Of Transcription ◽  
Domains Of Life ◽  
Sulfolobus Turreted Icosahedral Virus

AbstractRNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

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

1990 ◽  
Vol 270 (1) ◽  
pp. 141-148 ◽  
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.

scholarly journals Formation of Intermediate Transcription Initiation Complexes at pfliD and pflgM by ς28 RNA Polymerase

2001 ◽  
Vol 183 (21) ◽  
pp. 6244-6252 ◽  
Author(s):  
Jennifer R. Givens ◽  
Colleen L. McGovern ◽  
Alicia J. Dombroski
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
General Phenomenon ◽  
Temperature Dependent ◽  
Binding Properties ◽  
Serovar Typhimurium ◽  
Open Complex Formation ◽  
Flagellum Biosynthesis ◽  
Dna Strand

ABSTRACT The ς subunit of prokaryotic RNA polymerase is an important factor in the control of transcription initiation. Primary ς factors are essential for growth, while alternative ς factors are activated in response to various stimuli. Expression of class 3 genes during flagellum biosynthesis in Salmonella enterica serovar Typhimurium is dependent on the alternative ς factor ς28. Previously, a novel mechanism of transcription initiation at the fliC promoter by ς28holoenzyme was proposed. Here, we have characterized the mechanism of transcription initiation by a holoenzyme carrying ς28 at the fliD and flgM promoters to determine if the mechanism of initiation observed at pfliC is a general phenomenon for all ς28-dependent promoters. Temperature-dependent footprinting demonstrated that promoter binding properties and low-temperature open complex formation are similar for pfliC, pfliD, and pflgM. However, certain aspects of DNA strand separation and complex stability are promoter dependent. Open complexes form in a concerted manner at pflgM, while a sequential pattern of open complex formation occurs at pfliD. Open and initiated complexes formed by holoenzyme carrying ς28 are generally unstable to heparin challenge, with the exception of initiated complexes at pflgM, which are stable in the presence of nucleoside triphosphates.

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

1993 ◽  
Vol 289 (3) ◽  
pp. 771-775 ◽  
Author(s):  
S Minchin ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Detectable Effect ◽  
Guanine Residue ◽  
Transient Contact ◽  
Open Complex Formation ◽  
G 14 ◽  
Close Contacts

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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