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.

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.

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.

scholarly journals Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

2016 ◽  
Author(s):  
A. Ingargiola ◽  
E. Lerner ◽  
S. Chung ◽  
F. Panzeri ◽  
A. Gulinatti ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
High Throughput Analysis ◽  
Parallel Acquisition ◽  
Promoter Escape ◽  
Single Molecule Fret ◽  
Bacterial Rna ◽  
Measured Sample ◽  
Outstanding Question ◽  
Kinetics Of

AbstractWe describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription.Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

scholarly journals Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

2016 ◽  
Vol 113 (43) ◽  
pp. E6562-E6571 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung Lee ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Magnetic Tweezers ◽  
Nucleoside Triphosphate ◽  
Biologically Relevant ◽  
Rate Limiting Step ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP–GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused–backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

scholarly journals Structure of a bacterial RNA polymerase holoenzyme open promoter complex

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Brian Bae ◽  
Andrey Feklistov ◽  
Agnieszka Lass-Napiorkowska ◽  
Robert Landick ◽  
Seth A Darst
Keyword(s):  
Rna Polymerase ◽  
Bubble Formation ◽  
Thermus Aquaticus ◽  
Transcription Bubble ◽  
Template Strand ◽  
Bacterial Rna ◽  
Promoter Complex ◽  
Primary Means ◽  
Promoter Dna ◽  
Biochemical Analyses

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.

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