Biomedical subjects

M C J Wilce

Publications and source records attributed to M C J Wilce.

4 recordsLinked to original sources

PubMed2006

Crystallization and preliminary X-ray diffraction analysis of the Bacillus subtilis replication termination protein in complex with the 37-base-pair TerI-binding site.

The replication terminator protein (RTP) of Bacillus subtilis binds to specific DNA sequences that halt the progression of the replisome in a polar manner. These terminator complexes flank a defined region of the chromosome into which they allow replication forks to enter but not exit. Forcing the fusion of replication forks in a specific zone is thought to allow the coordination of post-replicative processes. The functional terminator complex comprises two homodimers each of 29 kDa bound to overlapping binding sites. A preparation of RTP and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. A data set to 3.9 A resolution with 97.0% completeness and an R(sym) of 12% was collected from a single flash-cooled crystal using synchrotron radiation. The diffraction data are consistent with space group P622, with unit-cell parameters a = b = 118.8, c = 142.6 A.

Bacillus subtilis↗

PubMed2005

Formation of an alphaCP1-KH3 complex with UC-rich RNA.

The alphaCP family of proteins [also known as poly(C)-binding or heterogeneous nuclear ribonucleoprotein E proteins] are involved in the regulation of messenger RNA (mRNA) stability and translational efficiency. They bind via their triple heterologous nuclear ribonucleoprotein K homology (KH) domain structures to C-rich mRNA, and are thought to interact with other mRNA-binding proteins as well as provide direct nuclease protection. In particular, alphaCP1 and alphaCP2 have been shown to bind to a specific region of androgen receptor (AR) mRNA, resulting in its increased stability. The roles of each of the KH motifs in the binding affinity and the specificity is not yet understood. We report the beginning of a systematic study of each of the alphaCP KH domains, with the cloning and expression of alphaCP1-KH2 and alphaCP1-KH3. We report the ability of alphaCP1-KH3, but not alphaCP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an alphaCP1-KH3/AR mRNA complex for structural studies. (1)H-(15)N heteronuclear single quantum correlation NMR spectra of (15)N-labelled alphaCP1-KH3 verified the integrity and good solution behaviour of the purified domain. The titration of the 11-nucleotide RNA target sequence from AR mRNA resulted in a rearrangement of the (1)H-(15)N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.

Amino Acid Motifs↗

PubMed2005

Structure and RNA binding of the third KH domain of poly(C)-binding protein 1.

Poly(C)-binding proteins (CPs) are important regulators of mRNA stability and translational regulation. They recognize C-rich RNA through their triple KH (hn RNP K homology) domain structures and are thought to carry out their function though direct protection of mRNA sites as well as through interactions with other RNA-binding proteins. We report the crystallographically derived structure of the third domain of alphaCP1 to 2.1 A resolution. alphaCP1-KH3 assumes a classical type I KH domain fold with a triple-stranded beta-sheet held against a three-helix cluster in a betaalphaalphabetabetaalpha configuration. Its binding affinity to an RNA sequence from the 3'-untranslated region (3'-UTR) of androgen receptor mRNA was determined using surface plasmon resonance, giving a K(d) of 4.37 microM, which is indicative of intermediate binding. A model of alphaCP1-KH3 with poly(C)-RNA was generated by homology to a recently reported RNA-bound KH domain structure and suggests the molecular basis for oligonucleotide binding and poly(C)-RNA specificity.

3' Untranslated Regions↗

PubMed2003

The impact of single cysteine residue mutations on the replication terminator protein.

We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies. The cysteine residue 110 was replaced with alanine, valine or serine. Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP. The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity. The structure of RTP.C110S, solved to 2.5A resolution using crystallographic methods, showed no major structural perturbation due to the mutation. Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation. The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.

Bacterial Proteins↗