ANALYSIS OF RNA CHAIN ELONGATION AND TERMINATION BY SACCHAROMYCES-CEREVISIAE RNA POLYMERASE-III

When Saccharomyces cerevisiae RNA polymerase (Pol) III transcribes the S. cerevisiae SUP4 tRNA(Tyr) gene, it is obliged to navigate past a large, multi-subunit DNA-bound complex of proteins. We have analyzed individual steps of RNA chain elongation on this gene. Slow steps of transcriptional initiation were by-passed by forming 5′-end-labeled arrested and precisely positioned transcription complexes. Synchronous resumption of chain elongation by these complexes allowed a single round of RNA synthesis and termination to be analyzed in detail. Results for synthesis at 20°C and 0°C, in the presence of 100 μM and 1 mM ribonucleoside triphosphates (NTPs) are presented. RNA chain elongation through assembled transcription complexes was uneven but relatively rapid: at 20°C with 1 mM NTPs, the fastest RNA chains elongated at an average rate of 29 nucleotides (nt)/second, and the median RNA chains elongated at 21 to 22 nt/second on average. These rates are comparable with a recent measurement of the average rate of chain elongation in vivo by Drosophila RNA polymerase II at 25°C. At 0°C, RNA chain elongation rates were, on average, approximately 30-fold slower. Quantitative analysis of the individual steps of RNA chain elongation showed that steps of adding U and A to U-terminated RNA chains tended to be relatively slow, and to be more strongly influenced by nucleotide concentration. Termination of transcription occurred in the sequence T7GT6 (in the non-template DNA strand) and was progressive. Transcripts with five, six and seven U residues were formed, and there was even slow readthrough of the T7 stretch, with GU3 adding rapidly, suggesting that incorporation of a single G into the RNA chain served to reset elongation rates substantially or entirely. Stripping transcription factor (TF) IIIC from transcription complexes did not substantially increase overall RNA chain growth rate, but did diminish pausing at a single site upstream of the boxB binding site of TFIIIC. The TFIIIC-generated delay at this single site was estimated to be only approximately 0.15 to 0.2 seconds at 20°C. Quantitative analysis of RNA chain elongation yielded kinetic parameters for the individual steps of nucleotide addition that were used in computer simulations of RNA chain growth. Elongation modeled as a simple sequence of pseudo-first-order reactions yielded computed RNA chain length distributions that remained relatively synchronous during elongation, while observed chain growth quickly became desynchronized. The discrepancy points to complexities of RNA chain elongation that are discussed. In particular, we explored the idea, that Pol III can switch repeatedly between rapidly stepping and slowly stepping states during the elongation of a single RNA molecule, by appropriate computer simulations. © 1994 Academic Press Limited.