title: An insertion in the catalytic trigger loop gates the secondary channel of RNA polymerase.
abstract: Escherichia coli DksA and GreB bind to RNA polymerase (RNAP), reaching inside the secondary channel, with similar affinities but have different cellular functions. DksA destabilizes promoter complexes whereas GreB facilitates RNA cleavage in arrested elongation complexes (ECs). Although the less abundant GreB may not interfere with DksA regulation during initiation, reports that DksA acts during elongation and termination suggest that it may exclude GreB from arrested complexes, potentially triggering genome instability. Here, we show that GreB does not compete with DksA during termination whereas DksA, even when present in several hundredfold molar excess, does not inhibit GreB-mediated cleavage of the nascent RNA. Our findings that DksA does not bind to backtracked or active ECs provide an explanation for the lack of DksA activity on most ECs that we reported previously, raising a question of what makes a transcription complex susceptible to DksA. Structural modeling suggests that i6, an insertion in the catalytic trigger loop, hinders DksA access into the channel, restricting DksA action to a subset of transcription complexes. In support of this hypothesis, we demonstrate that deletion of i6 permits DksA binding to ECs and that the distribution of DksA and i6 in bacterial genomes is strongly concordant. We hypothesize that DksA binds to transcription complexes in which i6 becomes mobile, for example, as a consequence of weakened RNAP interactions with the downstream duplex DNA.
authors.affiliation: Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Furman
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Tsodikov
authors.forename: Yuri I
authors.initials: YI
authors.lastname: Wolf
authors.forename: Irina
authors.initials: I
authors.lastname: Artsimovitch
mesh_terms: D000595:Amino Acid Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059367:RNA Cleavage; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D061805:Transcription Elongation, Genetic; D061806:Transcription Termination, Genetic; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C079667:GreB protein, E coli; D012329:RNA, Bacterial; D035602:Transcriptional Elongation Factors; C090751:dksA protein, E coli; D012321:DNA-Directed RNA Polymerases