Biomedical subjects

John P Richardson

Publications and source records attributed to John P Richardson.

5 recordsLinked to original sources

PubMed2006

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.

Base Sequence↗

PubMed2006

How Rho exerts its muscle on RNA.

Rho factor in bacteria terminates transcription by using energy from ATP hydrolysis to forcefully dissociate the transcripts from RNA polymerase. used data from presteady-state ATPase kinetics to support a rational mechanistic model for Rho's action on RNA.

Adenosine Triphosphatases↗

PubMed2005

Identification of a structural element that is essential for two functions of transcription factor NusG.

The transcription factor NusG from Escherichia coli modulates the rate of transcript elongation by RNA polymerase and the efficiency of Rho-dependent transcript termination. It consists of two globular domains with an extra loop extending out of the amino-terminal domain in the position that is occupied by a third globular domain in some NusG homologues. We have tested the role of this appended mini-domain by assaying the elongation and termination enhancement activities of variants. The results show that variants with changes in their sequence do not cause a loss of functions, whereas variants with the deletions of the residues in that domain are much less active for both functions. This finding suggests that the mini-domain serves as a structural element for an interaction rather than as a site for residue-specific contacts.

DNA-Directed RNA Polymerases↗

PubMed2003

Loading Rho to terminate transcription.

In bacteria, one of the major transcriptional termination mechanisms requires a hexameric RNA/DNA helicase known as Rho. One question that has remained unanswered is how the helicase loads onto a nascent transcript so that it can initiate actions on the transcript to cause termination. Recent structures of Rho bound to nucleic acid by show how the individual RNA-binding domains of the 6 subunits are organized and that the ring is split open. The opening is wide enough to accommodate single-stranded RNA and suggests that this conformation is poised to load onto mRNA.

Binding Sites↗

PubMed2002

Rho-dependent termination and ATPases in transcript termination.

Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA-binding domain (RNA-BD) of known structure as well as an ATP-binding domain (ATP-BD) for which a structure has been proposed based on homology modeling. A model is proposed in which Rho first makes an interaction with a nascent RNA on a C-rich, primarily single-stranded rut region of the transcript as that region emerges from the exit site of RNA polymerase. A subsequent step involves a temporary release of one subunit of the hexamer to allow the 3' segment of the nascent transcript to enter the central channel of the Rho ring. Actions of the Rho structure in the channel on the 3' segment that are coupled to ATP hydrolysis pull the RNA from its contacts with the template and RNA polymerase, thus causing termination of its synthesis.

Adenosine Triphosphatases↗