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Published on: August 26, 2012
Recognition of a human arrest site is conserved between RNA polymerase II and prokaryotic RNA polymerases
1Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Abstract:
DNA sequences that arrest transcription by either eukaryotic RNA polymerase II or Escherichia coli RNA polymerase have been identified previously. Elongation factors SII and GreB are RNA polymerase-binding proteins that enable readthrough of arrest sites by these enzymes, respectively. This functional similarity has led to general models of elongation applicable to both eukaryotic and prokaryotic enzymes. Here we have transcribed with phage and bacterial RNA polymerases, a human DNA sequence previously defined as an arrest site for RNA polymerase II. The phage and bacterial enzymes both respond efficiently to the arrest signal in vitro at limiting levels of nucleoside triphosphates. The E. coli polymerase remains in a template-engaged complex for many hours, can be isolated, and is potentially active. The enzyme displays a relatively slow first-order loss of elongation competence as it dwells at the arrest site. Bacterial RNA polymerase arrested at the human site is reactivated by GreB in the same way that RNA polymerase II arrested at this site is stimulated by SII. Very efficient readthrough can be achieved by phage, bacterial, and eukaryotic RNA polymerases in the absence of elongation factors if 5-Br-UTP is substituted for UTP. These findings provide additional and direct evidence for functional similarity between prokaryotic and eukaryotic transcription elongation and readthrough mechanisms.
Insights
Bacterial and phage RNA polymerases arrest at human DNA sequences, similar to eukaryotic RNA polymerase II. Elongation factors GreB and SII facilitate readthrough, supporting conserved transcription mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA sequences can arrest transcription by RNA polymerases.
- Elongation factors like SII (eukaryotic) and GreB (prokaryotic) help polymerases overcome these arrest sites.
- This suggests conserved mechanisms in transcription elongation.
Purpose of the Study:
- To investigate if phage and bacterial RNA polymerases respond to a human DNA arrest site identified for eukaryotic RNA polymerase II.
- To compare the functional similarity of elongation and readthrough mechanisms between prokaryotic and eukaryotic transcription.
Main Methods:
- In vitro transcription assays using phage and bacterial RNA polymerases with a human DNA arrest sequence.
- Analysis of polymerase behavior at arrest sites, including complex stability and elongation competence.
- Testing the effect of elongation factors (GreB, SII) and nucleotide analogs (5-Br-UTP) on readthrough.
Main Results:
- Phage and bacterial RNA polymerases efficiently arrest at the human DNA sequence in vitro, especially at low nucleoside triphosphate levels.
- Escherichia coli RNA polymerase forms stable, potentially active complexes at the arrest site, with slow loss of elongation competence.
- Bacterial RNA polymerase arrest is relieved by GreB, mirroring SII's effect on eukaryotic RNA polymerase II.
- Efficient readthrough by all tested polymerases is achieved using 5-Br-UTP instead of UTP, without elongation factors.
Conclusions:
- Provides direct evidence for functional similarity between prokaryotic and eukaryotic transcription elongation and readthrough.
- Supports the hypothesis of general models for transcription elongation applicable to both prokaryotic and eukaryotic systems.
- Highlights the conserved nature of RNA polymerase-DNA interactions and the role of elongation factors in overcoming transcriptional roadblocks.
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