Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences

Abstract

We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thi) operon of Escherichia coli. Fourteen sequence variants located within its d(G+C) stem-loop and d(A+T)-rich regions were studied. We found that the helix integrity in the stem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency. The sequence of the G+C stem-loop itself also influences termination. Moreover, the dA template stretch at the 3' end of the terminator plays a major role in termination efficiency, but base pairing between the A and U tract of the transcript does not From the studies using deletion variants and a series of mutants that alter the sequences immediately downstream from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modulated by downstream DNA sequences in a sequence-specific manner. The second base immediately following the poly(T) tract is crucial for determining the termination efficiency by chloroplast RNA polymerase, but not of the T7 or E.coli enzymes.

3'-Inverted repeats in plant mitochondrial mRNAs are processing signals rather than transcription terminators

“…In the latter, such stem–loop structures have also been found to act as mRNA processing signals rather than terminating transcription. However, in these in vitro tests, a residual influence on transcriptional pausing and release of the nascent RNA may be effected by these IR tracts since, in the Chlamydomonas chloroplast, rbcL and psaB IRs appear to terminate transcription in vivo (Blowers et al ., 1993) and bacterial terminators such as the thra structure are still a hindrance to a spinach chloroplast RNA polymerase (Chen et al ., 1995).…”

Effects of rho‐independent terminators on the expression of the prokaryotic β‐glucuronidase gene in tobacco

“…Previous studies have shown that some point variants in the G+C‐rich region of the thr attenuator decrease the transcription termination of E. coli (Lynn et al 1985, 1987, 1988; Burton 1989; Yang and Gardner 1989), bacteriophage (Jeng et al 1990, 1992, 1997), and chloroplast (Chen et al 1995) RNA polymerases. To analyze the effects of these altered thr attenuators on plant gene expression, variants containing the transcription‐termination structure of the thr operon regulatory site were cloned into the region between the 35S promoter and GUS coding region of pBI221SI to become a pBI221SIthr series (Fig.…”

“…The threonine ( thr ) attenuator contains a G+C‐rich dyad symmetry followed by a run of deoxythymidine, encodes an RNA hairpin with a stretch of uridine, and is from the regulatory region of the thr operon of E. coli . The effects of the thr attenuator on transcription termination have been well studied with E. coli (Yang and Gardner 1989), bacteriophage (Jeng et al 1990, 1992, 1997), and chloroplast (Chen et al 1995) RNA polymerases. Results from these studies indicate that the G:C base pairing in the stem region of the hairpin and a certain length of the polyuridine tract of the thr RNA hairpin are required for these RNA polymerases to terminate well.…”

Primary structure requirements for in vivo activity and bidirectional function of the transcription terminator shared by the oppositely oriented skc /rel-orf1 genes of Streptococcus equisimilis H46A

“…An early model for transcription termination, which focused on nucleic acid destabilization, proposes that the transcribed RNA forms a hairpin which causes RNA polymerase to pause, and unstable pairing of dA and rU residues between the template and the nascent RNA promotes the release of the transcript (Farnham and Platt 1980;Martin and Tinoco 1980;von Hippel 1987, 1991;Richardson 1993). Extended analyses of terminator mutations, including point substitutions in the (G + C)-rich region, inversion of loop sequences, and nested deletions in the oligo(dT) stretch, have yielded results that, in the main, are in conformity with the classical thermodynamic termination model (Lynn et al 1985(Lynn et al , 1988Jeng et al 1990;Cheng et al 1991;Chen et al 1995;Wilson and von Hippel 1995). However, several lines of evidence indicate that additional sequence elements can aect termination eciency.…”

“…However, several lines of evidence indicate that additional sequence elements can aect termination eciency. Among these, upstream sequences including regions close to the promoter, rA tracts just upstream of the RNA hairpin, and non-transcribed sequences downstream of the termination point are known to modulate the eciency of the termination process (Goliger et al 1989;Telesnitsky and Chamberlin 1989a, b;Lee et al 1990;Jeng et al 1990Jeng et al , 1992Wright et al 1992;Chen et al 1995). Based on such observations, as well as results showing that milieu conditions greatly in¯uence transcription termination in a manner inconsistent with the thermodynamic model ), an alternative model has been postulated which describes termination as a multi-step process in which RNA polymerase participates in the control of termination eciency , Chamberlin 1994, Nudler et al 1995.…”