A DNA restriction fragment with convergent SP6 and T7 promoters has undergone reaction with cisdiamminedichloroplatinum(II) (cis-DDP) and was then used as a template for RNA synthesis in vitro. The T7 and SP6 RNA polymerases generate fragments of defmed sizes. Analysis of the RNA fragments shows that the polymerases are mainly blocked at the level of the d(GG) and d(AG) sites and to a lesser extent at the level of the d(GC) sites. The adducts at the d(GC) sites are more resistant to cyanide ion attack than those at the major sites and are identified as interstrand cross-links. The formation of an interstrand cross-link between the N-7 atoms of two guanine residues at the d(GC) sites was further confirmed by chemical modifications.cis-Diamminedichloroplatinum(II) (cis-DDP) is an antitumor agent of major clinical importance. Much evidence suggests that the cytotoxic action of the drug is related to its ability to react with DNA even though the mechanism of action is still unknown (for general reviews, see refs. 1-5 and references therein). Like many chemicals used in cancer chemotherapy, cis-DDP is a bifunctional agent that forms in vivo and in vitro intrastrand and interstrand cross-links on DNA. The major lesions are d(GG) and d(AG) 1,2-intrastrand cross-links, representing 65% and 25% of the total adducts, respectively (1-5). Although the interstrand cross-links represent a minor portion (<10% of total adducts), several experiments in tissue culture systems have correlated the DNA interstrand cross-linking reaction with cytotoxicity (for general reviews, see refs. 6 and 7).DNA interstrand cross-linking occurs predominantly between two guanine N-7 atoms on opposite strands (2,8,9).A distance of -3 A is required for the cis-DDP cross-linking reaction (10, 11). Thus, two adjacent guanine residues on the opposite strands, either in the 5'-CG-3' or 5'-GC-3' sequences, are the most probable reaction sites on DNA. However, in both cases, formation of the cross-link in B-DNA implies a large distortion of the double helix since the two N-7 atoms in d(CG) and d(GC) sequences are separated by about 9 and 7 A (12), respectively. Recently, manipulation of three-dimensional molecular models inferred that the d(CG) sequence is more able to match the interstrand crosslinking requirement (2,8).In vitro studies have shown that DNA synthesis by DNA polymerases of different origins acting on cis-DDP-modified DNAs was arrested at the level of the adducts (1-5, 13-16). This finding has been extensively used to map the sites of platination on DNA. However, in these studies, the interstrand adducts were not detected because either the platinated DNA was single stranded or the assay was not sensitive enough.We show here that the in vitro RNA synthesis by bacteriophage RNA polymerase acting on platinated DNA is blocked at the level of the adducts. The ability of the adducts to terminate transcription has been used to map the cis-DDP binding sites on a double-stranded DNA. Furthermore, analysis of the modification patterns shows ...
Gene regulation by bacterial trans-encoded small RNAs (sRNAs) is generally regarded as a post-transcriptional process bearing exclusively on the translation and/or the stability of target messenger RNA (mRNA). The work presented here revealed the existence of a transcriptional component in the regulation of a bicistronic operon-the chiPQ locus-by the ChiX sRNA in Salmonella. By studying the mechanism by which ChiX, upon pairing near the 59 end of the transcript, represses the distal gene in the operon, we discovered that the action of the sRNA induces Rho-dependent transcription termination within the chiP cistron. Apparently, by inhibiting chiP mRNA translation cotranscriptionally, ChiX uncouples translation from transcription, causing the nascent mRNA to become susceptible to Rho action. A Rho utilization (rut) site was identified in vivo through mutational analysis, and the termination pattern was characterized in vitro with a purified system. Remarkably, Rho activity at this site was found to be completely dependent on the function of the NusG protein both in vivo and in vitro. The recognition that transencoded sRNA act cotranscriptionally unveils a hitherto neglected aspect of sRNA function in bacteria.
RNA-binding protein CsrA is a key regulator of a variety of cellular processes in bacteria, including carbon and stationary phase metabolism, biofilm formation, quorum sensing, and virulence gene expression in pathogens. CsrA binds to bipartite sequence elements at or near the ribosome loading site in messenger RNA (mRNA), most often inhibiting translation initiation. Here we describe an alternative novel mechanism through which CsrA achieves negative regulation. We show that CsrA binding to the upstream portion of the 59 untranslated region of Escherichia coli pgaA mRNA-encoding a polysaccharide adhesin export protein-unfolds a secondary structure that sequesters an entry site for transcription termination factor Rho, resulting in the premature stop of transcription. These findings establish a new paradigm for bacterial gene regulation in which remodeling of the nascent transcript by a regulatory protein promotes Rho-dependent transcription attenuation.
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