Sequence-specific interactions in the RNA-binding domain of Escherichia coli transcription termination factor Rho.
Rho factor is an essential protein that causes termination of transcription in a wide variety of bacteria by an RNA-dependent helicase activity. Rho is activated by transcripts that contain a high proportion of cytidine residues. The interaction between Rho and two adjacent cytidine residues within the bound RNA has been identified by previous crystallographic studies (Skordalakes, E., and Berger, J. M. (2003) Cell 114, 135-146). In this study, NMR methods were used to investigate the sequence dependence of the binding of oligonucleotides to the RNA-binding domain of Rho protein (rho130). A comparison of the NMR spectra obtained for rho130 bound to single-stranded oligonucleotides ACTTCCA or ATTTCCA showed that the 5'-cytidine residue interacts with Rho at a site that is distinct from the CC binding site identified by crystallographic studies. Two amino acid residues within this new cytidine binding site, Arg(88) and Phe(89), were altered to Glu and Ser, respectively. These mutant forms of Rho were defective in transcriptional termination, suggesting that those residues play an important role in the activation of Rho by bound RNA.
Hitchens
T Kevin
TK
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Zhan
Yiping
Y
Richardson
Lislott V
LV
Richardson
John P
JP
Rule
Gordon S
GS
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012234:Rho Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*
D012234:Rho Factor; D012313:RNA; D004247:DNA