Biomedical subjects

P B Rupert

Publications and source records attributed to P B Rupert.

6 recordsLinked to original sources

PubMed2002

The hairpin ribozyme: from crystal structure to function.

The hairpin ribozyme is one of four known natural catalytic RNAs that carry out sequence-specific cleavage of RNA. It is of particular biochemical interest because, unlike 'classic' ribozymes, such as the group I intron, it appears not to employ metal ions as catalytic cofactors. We have determined the crystal structure of a hairpin-ribozyme-inhibitor complex at a resolution of 2.4 A. The active site of the ribozyme results from docking of two irregular double helices. Docking results in major structural rearrangements of the helices, including a distortion of the substrate strand that brings it into a reactive conformation. It remains to be established whether this RNA enzyme relies exclusively on binding energy to carry out catalysis, or whether some other mechanism, such as general acid-base catalysis, is being employed.

Catalytic Domain↗

PubMed2001

Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis.

The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.

Base Sequence↗

PubMed2000

SRPrises in RNA-protein recognition.

The recent structure determination of the phylogenetically conserved cor re of the signal recognition particle (SRP) reveals a novel of highly un usual RNA-protein interface, where non-canonical base pairs play a centr al role. The structure shows how a helix-turn-helix motif can be employ ed to bind RNA and offers hints as to how the signal peptide is recogniz ed by the SRP.

Helix-Turn-Helix Motifs↗

PubMed2000

The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers and dimers.

It was previously shown that the Cro repressor from phage lambda, which is a dimer, can be converted into a stable monomer by a five-amino acid insertion. Phe58 is the key residue involved in this transition, switching from interactions which stabilize the dimer to those which stabilize the monomer. Structural studies, however, suggested that Phe58 did not penetrate into the core of the monomer as well as it did into the native dimer. This was strongly supported by the finding that certain core-repacking mutations, including in particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly, the same substitution also increased the stability of the native dimer. At the same time it decreased the affinity of the dimer for operator DNA. Here we describe the crystal structures of the Cro F58W mutant, both as the monomer and as the dimer. The F58W monomer crystallized in a form different from that of the original monomer. In contrast to that structure, which resembled the DNA-bound form of Cro, the F58W monomer is closer in structure to wild-type (i.e. non-bound) Cro. The F58W dimer also crystallizes in a form different from the native dimer but has a remarkably similar overall structure which tends to confirm the large changes in conformation of Cro on binding DNA. Introduction of Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact residue, providing a structural explanation for the reduction in DNA-binding affinity. The improved thermal stability is seen to be due to the enhanced solvent transfer free energy of Trp58 relative to Phe58, supplemented in the dimer structure, although not the monomer, by a reduction in volume of internal cavities.

Bacteriophage lambda↗

PubMed1998

A new DNA-binding motif in the Skn-1 binding domain-DNA complex.

The DNA-binding domain of Skn-1, a developmental transcription factor that specifies mesoderm in C. elegans, is shown by X-ray crystallography to have a novel fold in which a compact, monomeric, four-helix unit organizes two DNA-contact elements. At the C-terminus, a helix extends from the domain to occupy the major groove of DNA in a manner similar to bZip proteins. Skn-1, however, lacks the leucine zipper found in all bZips. Additional contacts with the DNA are made by a short basic segment at the N-terminus of the domain, reminiscent of the 'homeodomain arm'.

Amino Acid Sequence↗

PubMed1994

Signal transduction in chemotaxis. A propagating conformation change upon phosphorylation of CheY.

The CheY protein from Escherichia coli and Salmonella typhimurium are among the best characterized proteins of the receiver domain family of two component signal transduction systems in bacteria. Phosphorylation of CheY plays a central role in bacterial chemotaxis. However, it is not entirely clear how its state of phosphorylation contributes to its function. Genetic evidence suggests that CheY changes its conformation upon phosphorylation. We present evidence for this conformation change by comparing the NMR 15N-1H correlation spectra of CheY.Mg2+ complex and phospho-CheY in the presence of magnesium. Large changes in chemical shift are used as indicators of chemical changes and probable structural changes in the protein backbone. Our observations suggest that significant structural changes occur in CheY upon phosphorylation and that these changes are distinct from the changes produced by magnesium ion binding. In addition to residues Asn-59 and Gly-65 that are immediately adjacent to the site of phosphorylation at Asp-57, a large number of other residues show significant chemical shift changes as a result of phosphorylation. These include Met-17, Val-21, Asn-23, Gly-39, Met-60, Met-63, Asp-64, Leu-66, Glu-67, Leu-68, Leu-69, Met-85, Val-86, Thr-87, Ala-88, Asn-94, Val-107, Lys-109, Thr-112, Ala-113, Ala-114, and Asn-121. These results appear inconsistent with the recent suggestion that phosphorylation produces the same structural changes as magnesium binding (Bellsolell, L., Prieto, J., Serrano, L., and Coll, M. (1994) J. Mol. Biol. 238, 489-495). We find that some regions change overlap with a genetically defined motor binding face. We therefore propose that the conformation switch modulates the interaction of CheY with its target, the flagellar motor. Other regions also change, possibly reflecting the many different functions of CheY homologues.

Amino Acid Sequence↗