Abstract
The kinetics of elongation of the mRNA that initiates from the lambda PR promoter has been examined using specific restriction fragments as template, and the locations at which significant pausing of the RNA polymerase occurs during in vitro transcription have been established. Major pausing of polymerase, in the absence of rho, occurs at the five rho-dependent termination sites (located between 290 and 450 base pairs downstream from PR) that are described in the accompanying article (Morgan, W. D., Bear, D. G., and von Hippel, P. H. (1983) J. Biol. Chem. 258, 9553-9564). The replacement of guanosine by inosine triphosphate in the transcription mix results in the appearance of new pausing sites; these pausing sites correspond, in part, to the new rho-dependent termini between 100 and 260 base pairs from PR identified in the preceding article (cited above) when inosine replaces guanosine in the transcript. The effects of variations in nucleoside triphosphate and salt concentrations on pausing have also been determined. Analysis of the base pair sequences of pausing sites shows that pausing may result from the presence of dyad symmetry, GC-rich sequences, or (for inosine-substituted transcripts) C-rich sequences in the RNA-DNA hybrid region. Quantitation of RNA polymerase pausing at termination loci indicates that pausing sites with relaxation times of 10 to 25 s (at 37 degrees C and 100 to 200 mM KCl) can lead to significant rho-dependent termination. In addition, increasing the length of "natural" pauses by lowering the concentrations of specific nucleoside triphosphate substrates can lead to increased termination efficiency, but only at sites that correspond to rho-dependent termini in elongation experiments conducted at standard concentrations of nucleoside triphosphates. These results, and the findings of the article cited above, are interpreted in terms of a two-component model for rho-dependent termination. Required are: (i) a significant pause in transcript elongation due to sequence and/or structural features at the termination site(s); and (ii) a rho-binding site(s) on the nascent mRNA that is long (70-90 nucleotide residues) and relatively free of secondary structure, and that contains appropriate sequences of cytidine residues.
Highlights
The kineticsof elongation of the mRNA that initiates structural features at the termination site(s); a(niid)a from the X PRpromoter has been examined using spe- rho-binding site(s) on the nascent mRNA that is long cific restriction fragments as template, and the loca- (70-90 nucleotide residues) and relativelyfree of sections at which significant pausing of the RNA polym- ondary structure, and that contains appropriate seerase occurs during in vitro transcription have been quences of cytidine residues
The replacement of guanosine by inosine triphosphate gether with work from other laboratories, showed that these in the transcription mix results in the appearance of termination loci are heterogeneous in detailed sequence and new pausing sites; these pausing sites correspond, in potential secondary structure, the termini appeared part, to the new rho-dependent termini between 100 to have certaingeneral features in common (includinga fairly and 260 base pairs fromPRidentified in the precedingstablepenultimatestem-loopstructureandperhapsother article when inosine replaces guanosine aspects; see Ref. 1, Fig. 8)
The effoefcvtsariations innucleoside rho-dependent termini could be established by replacing triphosphate and salt concentrationson pausing have guanosine residues with inosine in the nascent mRNA tranalso been determined
Summary
The kineticsof elongation of the mRNA that initiates structural features at the termination site(s); a(niid)a from the X PRpromoter has been examined using spe- rho-binding site(s) on the nascent mRNA that is long cific restriction fragments as template, and the loca- (70-90 nucleotide residues) and relativelyfree of sections at which significant pausing of the RNA polym- ondary structure, and that contains appropriate seerase occurs during in vitro transcription have been quences of cytidine residues. N addition, increasing the lengtohf %atural” pauses by lowering the concentrations of specific nucleoside triphosphate subrelatively specific rhobindingto a discretesite(s)onthe nascentmRNA, was found to be needed to definea rhodependent termination site aswell In this paperw, e examine the kineticosf RNA polymerasecatalyzed mRNA elongation during transcription from the h PKpromoter. We define the sequences of the sites at which strates can lead to increased termination efficiency, polymerase pausing occurs, anddeterminetherelaxation but only at sites that correspond rthoo-dependent ter- times for continued elongation through some of these pausing mini in elongation experiments conductedat standard sites. The relationship between these pausing sites and the concentrations of nucleoside triphosphates. Required are: (i) a significant the mechanismof rho-dependent termination
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