In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 100, No. 16 ( 2003-08-05), p. 9250-9255
Abstract:
Coliphage N4 virion RNA polymerase (vRNAP), the most distantly related
member of the T7-like family of RNA polymerases, is responsible for transcription of the early genes of the linear double-stranded DNA phage
genome. Escherichia coli single-stranded DNA-binding protein
( Eco SSB) is required for N4 early transcription in vivo , as
well as for in vitro transcription on super-coiled DNA templates
containing vRNAP promoters. In contrast to other DNA-dependent RNA polymerases, vRNAP initiates transcription on single-stranded,
promoter-containing templates with in vivo specificity; however, the
RNA product is not displaced, thus limiting template usage to one round. We show that Eco SSB activates vRNAP transcription at limiting
single-stranded template concentrations through template recycling. Eco SSB binds to the template and to the nascent transcript and
prevents the formation of a transcriptionally inert RNA:DNA hybrid. Using C-terminally truncated Eco SSB mutant proteins, human mitochondrial
SSB ( Hs mt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hs mt- Eco SSB chimera, we have mapped a determinant of
template recycling to the C-terminal amino acids of Eco SSB. T7 RNAP
contains an amino-terminal domain responsible for binding the RNA product as it exits from the enzyme. No sequence similarity to this domain exists in
vRNAP. Hereby, we propose a unique role for Eco SSB: It functionally
substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1133325100
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2003
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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