Opposite Consequences of Two Transcription Pauses Caused by an Intrinsic Terminator Oligo(U)

Abstract

The RNA oligo(U) sequence, along with an immediately preceding RNA hairpin structure, is an essential cis-acting element for bacterial class I intrinsic termination. This sequence not only causes a pause in transcription during the beginning of the termination process but also facilitates transcript release at the end of the process. In this study, the oligo(U) sequence of the bacteriophage T7 intrinsic terminator T, rather than the hairpin structure, induced pauses of phage T7 RNA polymerase not only at the termination site, triggering a termination process, but also 3 bp upstream, exerting an antitermination effect. The upstream pause presumably allowed RNA to form a thermodynamically more stable secondary structure rather than a terminator hairpin and to persist because the 5-half of the terminator hairpin-forming sequence could be sequestered by a farther upstream sequence via sequence-specific hybridization, prohibiting formation of the terminator hairpin and termination. The putative antiterminator RNA structure lacked several base pairs essential for termination when probed using RNases A, T1, and V1. When the antiterminator was destabilized by incorporation of IMP into nascent RNA at G residue positions, antitermination was abolished. Furthermore, antitermination strength increased with more stable antiterminator secondary structures and longer pauses. Thus, the oligo(U)-mediated pause prior to the termination site can exert a cis-acting antitermination activity on intrinsic terminator T, and the termination efficiency depends primarily on the termination-interfering pause that precedes the termination-facilitating pause at the termination site.Multisubunit bacterial RNA polymerase transcription terminates upon various termination signals, causing disassembly of elongation complexes (ECs) 2 with or without the assistance of termination factors (1, 2). The most common class of intrinsic (factor-independent) termination signal relies on an oligo(U) sequence in the RNA preceded by an RNA hairpin structure (class I intrinsic termination signal) (3). The termination mechanism of class I signals is shared by virtually all multisubunit bacterial and single-subunit phage RNA polymerases. However, it is distinct from the mechanism of class II intrinsic termination, which is caused by specific DNA sequence recognition of single-subunit phage T7 RNA polymerase (4).The oligo(U) sequence allows ECs to pause prior to the release of RNA transcripts (5, 6). This transcription pause has been shown to be the first signal for the class I termination pathway, providing additional time for the formation of a terminator RNA hairpin that opens or unwinds the RNA-DNA hybrid at an upstream region, shortening and weakening the hybrid (5,7,8). Because the remaining downstream part of the hybridized region consists mostly of rU:dA base pairs, it becomes thermodynamically unstable, facilitating the release of nascent RNA transcripts (9).In this study, an opposite effect of the oligo(U)-mediated pause was found with a typical class I...

Borrelia burgdorferi 297 bmpA encode the mRNA that contains ORF for a leader peptide that regulates bmpA gene expression.

“…In addition to a global regulatory system, prokaryotes can employ non-global mechanisms of virulence gene regulation. They include expression of non-coding RNAs [12,13], effects on mRNA secondary structure that forms terminator/antiterminator structure [14][15][16] and affects mRNA stability [17] as well as the differential efficiency of ribosomal binding [18,19].…”