Rho-dependent termination of transcription. II. Kinetics of mRNA elongation during transcription from the bacteriophage lambda PR promoter.

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.