During transcription of protein-coding genes, bacterial RNA polymerase (RNAP) is closely followed by a ribosome that is engaged in translation of the newly synthesized transcript. Here we show that as a result of translation-transcription coupling the overall elongation rate of transcription is tightly controlled by translation. Acceleration and deceleration of a ribosome results in corresponding changes in the speed of RNAP. Consistently, we found an inverse correlation between the number of rare codons in a gene, which delay ribosome progression, and the rate of transcription. We further show that the stimulating effect of a ribosome on RNAP is achieved by preventing RNAP from adopting non-productive states. The moving ribosome inhibits spontaneous backtracking of RNAP, thereby enhancing its pace and also facilitating read-through of roadblocks in vivo. Such a cooperative mechanism ensures the two gene expression machineries match precisely each other rates, so that the transcriptional yield is always adjusted to translational needs at different genes and under various growth conditions.

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 ...

Biological processes such as transcription may generate domains of supercoiling on a circular DNA. The existence of these domains in Escherichia coli was investigated by the ability of different lengths of (CG) tracts, cloned upstream or downstream from the tetracycline resistance gene (tet) of pBR322, to adopt the Z structure in vivo. Segments as short as 12 base pairs adopt the Z form when cloned upstream from the tet gene (Eco RI site), whereas no Z DNA was detected when this sequence was cloned downstream (Sty I site), even with a 74-base pair (CG) tract that requires less supercoiling than shorter tracts for the B-Z transition. Hence the localized supercoil density in pBR322 can be as high as -0.038 and as low as -0.021 at different loci. These data demonstrate the existence of the Z structure for commonly found natural sequences and support the notion of domains of negative supercoiling in vivo.

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