PMID:24853501

Citation	Vitiello, CL, Kireeva, ML, Lubkowska, L, Kashlev, M and Gottesman, M (2014) Coliphage HK022 Nun protein inhibits RNA polymerase translocation. Proc. Natl. Acad. Sci. U.S.A. 111:E2368-75
Abstract	The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β' subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5' edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.
Links	PubMed PMC4060646 Online version:10.1073/pnas.1319740111
Keywords	Bacteriophage lambda/genetics; Bacteriophage lambda/metabolism; DNA, Viral/chemistry; DNA, Viral/genetics; DNA-Directed RNA Polymerases/chemistry; DNA-Directed RNA Polymerases/genetics; DNA-Directed RNA Polymerases/metabolism; Diphosphates/metabolism; Models, Genetic; Models, Molecular; Mutation; Nucleic Acid Conformation; Nucleotides/genetics; Nucleotides/metabolism; Protein Binding; Protein Structure, Tertiary; RNA, Viral/chemistry; RNA, Viral/genetics; Templates, Genetic; Transcription Elongation, Genetic; Transcription Factors/chemistry; Transcription Factors/genetics; Transcription Factors/metabolism; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPHK0:VNUN		GO:0032785: negative regulation of DNA-templated transcription, elongation	ECO:0000314:		P		Figure 1B shows an assay showing the TEC complex formation time lapsed. The assay shows that in the wild type subjects there was an increased rate of pausing is occurring when compared to the knockout phenotype. Additionally there was shown to be RNAP arrest which can be observed in comparing lanes 3 and 6. This together shows that nun is regulating transcription elongation.	complete CACAO 13279

References

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