The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3 untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3 UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3 UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3 UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3 UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.

A number of new ruthenium compounds have been synthesised, isolated and characterised, which exhibit excellent cytotoxicity against a number of different human tumour cell lines including a defined cisplatin resistant cell line and colon cancer cell lines. Addition of hydrophobic groups to the ruthenium molecules has a positive effect on the cytotoxicity values. Evidence is provided that, after incubation of a ruthenium compound with a 46 mer oligonucleotide duplex and subsequent nuclease treatment, ruthenium is bound to a guanine residue.

Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of Escherichia coli RNA polymerase ␣ subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to ␣. Each CRP dimer was found to direct the positioning of one of the two ␣ subunit CTDs. Thus, the function of RNA polymerase may be subject to regulation through protein-protein interactions between the two ␣ subunits and two different species of transcription factors.The RNA polymerase holoenzyme of Escherichia coli is composed of core enzyme with subunit structure ␣ 2 ␤␤Ј, responsible for RNA polymerization, and one of multiple species of subunit, responsible for promoter recognition. Promoter selectivity of the holoenzyme is modulated by direct or indirect interaction with many transcription factors, resulting in switching of the global pattern of gene transcription according to the environment. The best-characterized target on the RNA polymerase involved in molecular communication with transcription factors is the ␣ subunit carboxy-terminal domain (CTD) that contains the contact sites for class I transcription factors. The ␣ subunit, consisting of 329 amino acid residues, is composed of two structural domains, each responsible for distinct functions (1-3) and each forming independent structural domains connected by a protease-sensitive flexible linker (4-6). The amino (N)-terminal domain from residues 20 to 235 plays a key role in RNA polymerase assembly by providing the contact surface for ␣ dimerization and binding of ␤ and ␤Ј subunits (7-10), whereas the CTD from residues 235 to 329 plays a regulatory role by providing the contact surfaces for trans-acting protein factors and cis-acting DNA elements (11)(12)(13)(14).Whereas the regulation of many E. coli promoters involves a single factor, some promoters are regulated by two or more transcription factors, and such coregulation systems involving multiple species of transcription factors can couple gene expression to diverse environmental conditions. Knowledge of the molecular mechanism of prokaryotic transcription regulation involving more than two factors would contribute much to understanding of the events carried out in eukaryotes, because the regulation of gene transcription in eukaryotes generally involves the action of multiple transcription factors. To gain insight into this problem, we analyzed interactions between RNA polymerase and cAMP receptor protein (CRP) dimers on promoters carrying tandem CRP-binding sites at various positions relative to the transcription start site. A set of promoters was constructed carrying one DNA site for CRP centered at position Ϫ41.5 upstream from the transcription start point and a second DNA site for CRP located further upstream (refs. 15