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Related Concept Videos

Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

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Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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The DNA replication, transcription, and translation processes are intricately coupled in bacteria, allowing efficient gene expression and rapid protein synthesis. While this physical and functional coordination is advantageous, it introduces challenges that bacteria overcome through specific regulatory mechanisms.Coupling of Replication, Transcription, and TranslationThe coupling of replication, transcription, and translation is a hallmark of bacterial gene expression. As the replisome unwinds...
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Bacterial Transcription01:53

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RNA polymerase (RNAP) carries out DNA-dependent RNA synthesis in both bacteria and eukaryotes. Bacteria do not have a membrane-bound nucleus. So, transcription and translation occur simultaneously, on the same DNA template.
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DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
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Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
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Real-Time Observation of Backtracking by Bacterial RNA Polymerase.

Agnieszka Lass-Napiorkowska1, Tomasz Heyduk1

  • 1Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine , Saint Louis, Missouri 63104, United States.

Biochemistry
|January 9, 2016
PubMed
Summary
This summary is machine-generated.

Bacterial RNA polymerase (RNAP) backtracking, a crucial enzyme movement, was observed in real-time using a novel fluorescence method. This technique revealed how nucleotide binding and DNA template structure influence RNAP backtracking dynamics.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Microbiology

Background:

  • RNA polymerase (RNAP) backtracking, the backward sliding of the enzyme along DNA and RNA, is vital for bacterial and eukaryotic cellular processes.
  • Understanding RNAP backtracking mechanisms is essential for deciphering gene expression regulation and cellular responses to DNA damage.

Purpose of the Study:

  • To develop and apply a novel fluorescence-based method for real-time observation of bacterial RNAP backtracking.
  • To investigate the influence of nucleotide binding and DNA template properties on RNAP backtracking kinetics and extent.

Main Methods:

  • A fluorescence-based approach utilizing a Cy3 probe incorporated into the non-template DNA strand near the backtracking site.
  • Real-time monitoring of RNAP proximity-induced fluorescence enhancement to track enzyme movement.
  • Experimental manipulation of nucleotide binding and DNA template integrity (abasic sites, neutravidin obstruction).

Main Results:

  • NTP binding to the RNAP active site inhibits backtracking, confirming the coupling of nucleotide binding to enzyme translocation.
  • Backtracking extent and kinetics are not solely dependent on DNA-RNA hybrid instability, suggesting complex sequence-dependent regulation.
  • RNAP backtracking plays a critical role in how the enzyme navigates and responds to obstacles within the DNA template.

Conclusions:

  • The developed fluorescence assay provides a powerful tool for real-time visualization and mechanistic studies of RNAP backtracking.
  • This study elucidates key factors, including NTP binding and DNA template sequence, that regulate RNAP backtracking.
  • The findings highlight the significance of RNAP backtracking in enzyme processivity and response to DNA template lesions.