Interplay between σ region 3.2 and secondary channel factors during promoter escape by bacterial RNA polymerase

2017 ◽  
Vol 474 (24) ◽  
pp. 4053-4064 ◽  
Author(s):  
Ivan Petushkov ◽  
Daria Esyunina ◽  
Vladimir Mekler ◽  
Konstantin Severinov ◽  
Danil Pupov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Molecular Beacon ◽  
Secondary Channel ◽  
Enzyme Active Site ◽  
Promoter Escape ◽  
Bacterial Rna ◽  
Transcription Complexes ◽  
Kinetics Of ◽  
Rnap Binding

In bacterial RNA polymerase (RNAP), conserved region 3.2 of the σ subunit was proposed to contribute to promoter escape by interacting with the 5′-end of nascent RNA, thus facilitating σ dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled σ70 subunit of Escherichia coli RNAP. We show that substitutions and deletions in σ region 3.2 decrease the rate of promoter escape and lead to accumulation of inactive complexes during transcription initiation. Secondary channel factors differentially regulate this process depending on the promoter and mutations in σ region 3.2. GreA generally increase the rate of promoter escape; DksA also stimulates promoter escape on certain templates, while GreB either stimulates or inhibits this process depending on the template. When observed, the stimulation of promoter escape correlates with the accumulation of stressed transcription complexes with scrunched DNA, while changes in the RNA 5′-end structure modulate promoter clearance. Thus, the initiation-to-elongation transition is controlled by a complex interplay between RNAP-binding protein factors and the growing RNA chain.

Kinetics of promoter escape by bacterial RNA polymerase: effects of promoter contacts and transcription bubble collapse

2014 ◽  
Vol 463 (1) ◽  
pp. 135-144 ◽  
Author(s):  
Je Ko ◽  
Tomasz Heyduk
Keyword(s):  
Rna Polymerase ◽  
Major Determinant ◽  
Bubble Collapse ◽  
Transcription Bubble ◽  
Promoter Escape ◽  
Bacterial Rna ◽  
Rate Limiting ◽  
Kinetics Of

Promoter escape by RNA polymerase is often rate-limiting in transcription of a gene. Our data show that the energy released from transcription bubble collapse accompanying the escape is not a major determinant of the kinetics of promoter escape.

scholarly journals Universal functions of the σ finger in alternative σ factors during transcription initiation by bacterial RNA polymerase

RNA Biology ◽  
2021 ◽  
pp. 1-10
Author(s):  
Anastasiya Oguienko ◽  
Ivan Petushkov ◽  
Danil Pupov ◽  
Daria Esyunina ◽  
Andrey Kulbachinskiy
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Universal Functions ◽  
Bacterial Rna

scholarly journals Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

2003 ◽  
Vol 50 (4) ◽  
pp. 909-920 ◽  
Author(s):  
Iwona K Kolasa ◽  
Tomasz Łoziński ◽  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Specific Interaction ◽  
Structural Data ◽  
Structural Morphology ◽  
Promoter Dna ◽  
Sigma Subunit ◽  
Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

scholarly journals Sigma factor 1 in chloroplast gene transcription and photosynthetic light acclimation

2019 ◽  
Vol 71 (3) ◽  
pp. 1029-1038 ◽  
Author(s):  
Lauren A Macadlo ◽  
Iskander M Ibrahim ◽  
Sujith Puthiyaveetil
Keyword(s):  
Rna Polymerase ◽  
Gene Transcription ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Sigma Factors ◽  
Transcriptional Level ◽  
Gene Promoters ◽  
Chloroplast Genes ◽  
Specific Subset ◽  
Bacterial Rna

Abstract Sigma factors are dissociable subunits of bacterial RNA polymerase that ensure efficient transcription initiation from gene promoters. Owing to their prokaryotic origin, chloroplasts possess a typical bacterial RNA polymerase together with its sigma factor subunit. The higher plant Arabidopsis thaliana contain as many as six sigma factors for the hundred or so of its chloroplast genes. The role of this relatively large number of transcription initiation factors for the miniature chloroplast genome, however, is not fully understood. Using two Arabidopsis T-DNA insertion mutants, we show that sigma factor 1 (SIG1) initiates transcription of a specific subset of chloroplast genes. We further show that the photosynthetic control of PSI reaction center gene transcription requires complementary regulation of the nuclear SIG1 gene at the transcriptional level. This SIG1 gene regulation is dependent on both a plastid redox signal and a light signal transduced by the phytochrome photoreceptor.

scholarly journals Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation

2018 ◽  
Vol 70 (6) ◽  
pp. 1111-1120.e3 ◽  
Author(s):  
Robert Glyde ◽  
Fuzhou Ye ◽  
Milija Jovanovic ◽  
Ioly Kotta-Loizou ◽  
Martin Buck ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Bacterial Rna

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 Bacterial RNA polymerase can retain σ70 throughout transcription

2016 ◽  
Vol 113 (3) ◽  
pp. 602-607 ◽  
Author(s):  
Timothy T. Harden ◽  
Christopher D. Wells ◽  
Larry J. Friedman ◽  
Robert Landick ◽  
Ann Hochschild ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Messenger Rna ◽  
Initiation Factors ◽  
Bacterial Rna ◽  
Direct Measurements ◽  
Promoter Recognition ◽  
Σ Factor ◽  
Transcript Elongation

Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ70, can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ70 retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ70–RNAP complex during initiation from the λ PR′ promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ70 and the kinetics of σ70 release from actively transcribing complexes. σ70 release from mature elongation complexes was slow (0.0038 s−1); a substantial subpopulation of elongation complexes retained σ70 throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ70 manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ70 during transcription.

scholarly journals A Novel Initiation Pathway in Escherichia Coli Transcription

2016 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Magnetic Tweezer ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

AbstractInitiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance:Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

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