Termination of transcription by Escherichia coli RNA polymerase: Influence of secondary structure of RNA transcripts on ρ-independent and ρ-dependent termination

Publisher Website
Google Scholar
Abstract
The effect of RNA secondary structure on ρ-independent and ρ-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine → hypoxanthine substitution, with attendant weakening of secondary structure, abolished the ρ-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. ρ-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major ρ-dependent termination sites on the T3 DNA—at 8 and 15%. The termination activity of ρ in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of ρ action but ρ was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased ρ-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated ρ-mediated ATP hydrolysis. The ρ-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive ρ isolated from rho 15 mutant of E. coli , which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G·C-rich regions in RNA stem-loop structures or RNA·DNA hybrids are essential for ρ-independent termination, whereas ρ-dependent termination requires weakly paired cytosine residues for its action.

This publication has 26 references indexed in Scilit: