NASCENT RNA IN TRANSCRIPTION COMPLEXES INTERACTS WITH CSPE, A SMALL PROTEIN IN ESCHERICHIA-COLI IMPLICATED IN CHROMATIN CONDENSATION

Proteins in a partially fractionated Escherichia coli extract that int eract with the nascent RNA in active transcription complexes from seve ral promoters were detected using the photocrosslinking ribonucleotide analogs 5-(azidophenacyl)thio-UTP or 5-(azidophenacyl)thio-CTP as tra nscription substrates. Upon irradiation of ternary transcription compl exes, several extract proteins were crosslinked to the RNA. Most notab ly, a small protein was crosslinked to the RNA in complexes on seven o f nine templates tested. This protein was purified and sequenced and f ound to match a hypothetical protein, MsmC/CspE, recently shown to be involved in chromatin partitioning. CspE has 69% amino acid sequence i dentity with the major cold shock protein in E, coli, CspA, which has been shown to bind to a DNA sequence designated the Y box, with the se quence 5'-ATTGG. Of the nine templates tested, CspE was found to be mo st heavily crosslinked to RNA from the lambda P-R' promoter, which is modified by the Q antiterminator protein. CspE was very heavily crossl inked to RNA only ten nucleotides long in initial ternary complexes on this promoter, but not to this same RNA after it had been released fr om the transcription complex. However, even when present from the star t of transcription, CspE did not crosslink to the RNA 82 nucleotides l ong in elongation complexes from this same promoter. Despite the loss of interaction with the RNA after polymerase had left the promoter, Cs pE inhibited Q-mediated transcriptional antitermination from P-R' in v itro almost 200 nucleotides downstream from the promoter, presumably b y interaction with the Y box DNA upstream from P-R', which overlaps wi th the binding site for the Q. A potential role for CspE and transcrip tion in chromosome condensation and nucleoid structure is discussed. ( C) 1998 Academic Press.