Isolation of small RNA-binding proteins from E. coli: evidence for frequent interaction of RNAs with RNA polymerase.
Bacterial small RNAs (sRNAs) are non-coding RNAs that regulate gene expression enabling cells to adapt to various growth conditions. Assuming that most RNAs require proteins to exert their activities, we purified and identified sRNA-binding factors via affinity chromatography and mass spectrometry. We consistently obtained RNA polymerase betasubunit, host factor Hfq and ribosomal protein S1 as sRNA-binding proteins in addition to several other factors. Most importantly, we observed that RNA polymerase not only binds several sRNAs but also reacts with them, both cleaving and extending the RNAs at their 3' ends. The fact that the RNA polymerase reacts with sRNAs maps their interaction site to the active centre cleft of the enzyme and shows that it takes RNAs as template to perform RNA-dependent RNA polymerase activity. We further performed genomic SELEX to isolate RNA polymerase-binding RNAs and obtained a large number of E. coli sequences binding with high affinity to this enzyme. In vivo binding of some of the RNAs to the RNA polymerase was confirmed via co-immunoprecipitation in cell extracts prepared from different growth conditions. Our observations show that RNA polymerase is able to bind and react with many different RNAs and we suggest that RNAs are involved in transcriptional regulation more frequently than anticipated.
Windbichler
Nikolai
N
Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
von Pelchrzim
Frederike
F
Mayer
Oliver
O
Csaszar
Edina
E
Schroeder
Renée
R
D020413:3' Untranslated Regions; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*
D020413:3' Untranslated Regions; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit