Polymerase Elongation Rate
Even more than in DNA synthesis, it is sensible for cells to regulate RNA synthesis at the initiation steps so that the elaborate machinery involved in independently regulating thousands of genes need not be built into the basic RNA synthesis module. Once RNA synthesis has been initiated, it proceeds at the same average rate on most, independent of growth conditions. Can this be demonstrated? Another need for knowing the RNA elongation rate is in the interpretation of physiological experiments. How soon after the addition of an inducer can a newly synthe-sized mRNA molecule appear?
RNA elongation rate measurements are not too hard to perform invitro, but they are appreciably more difficult to perform on growing cells. Here we shall explain one method that has been used to determine the in vivo RNA elongation rate in Escherichia coli.
Figure 5.1 Effects of rifamycin addition on transcription of a large operon.Upon the addition of rifamycin, no more RNA polymerase molecules may initiate transcription. Those polymerase molecules that were transcribing con-tinue to the end of the operon. Finally, the polymerase molecule that had initiated transcription just before the addition of rifamycin completes tran-scription of the operon.
The measurement used rifamycin, an antibiotic that inhibits RNA polymerase only at the initiation step. It has no effect on RNA polym-erase molecules engaged in elongation. Rifamycin and radioactive urid-ine were simultaneously added to bacteria; thus only those RNA chains that were in the process of elongation at the time of the additions were radioactively labeled, and no new ones could be initiated (Fig. 5.1). At various times after the rifamycin and uridine addition, samples were taken from the culture and their RNA was separated according to size
Figure 5.2 Structure of the ribosomal RNA operon used to determine the RNAelongation rate in E. coli.
by electrophoresis on polyacrylamide gels. Suppose that a specific species of RNA molecule is well separated from all other species by the electrophoresis. Then, the radioactivity in this size class will increase with time for as long as RNA polymerase molecules transcribe the corresponding gene, but once the last polymerase molecule to initiate has crossed the region, there can be no additional increase in radioac-tivity. The interval between the addition of rifamycin and the end of the period over which radioactivity increases is the time required for an RNA polymerase molecule to transcribe from the promoter to the end of the transcribed region.
The ribosomal RNA gene complexes were a convenient system for these measurements. Each of these seven nearly identical gene com-plexes consists of two closely spaced promoters, a gene for the 16S ribosomal RNA, a spacer region, a tRNA gene, the gene for the 23S ribosomal RNA, and the gene for the 5S ribosomal RNA (Fig. 5.2). The total length of this transcriptional unit is about 5,000 nucleotides. The 16S RNA, spacer tRNA, 23S RNA, and 5S RNA are all generated by cleavage from the growing polynucleotide chain.
The interval between the time of rifamycin addition and the time at which the last RNA polymerase molecule transcribes across the end of the 5S gene is the time required for RNA polymerase to transcribe the 5,000 bases from the promoter to the end of the ribosomal gene com-plex. This time is found from the radioactive uridine incorporation measurements. Transcription across the 5S gene ends when the radio - activity in 5S RNA stops increasing. This happens about 90 seconds after rifamycin and uridine addition (Fig. 5.3). This yields an elongation rate of about 60 nucleotides per second. This type of elongation rate meas-urement has been performed on cells growing at many different growth rates, and as expected, the results show that the RNA chain growth rate is independent of the growth rate of cells at a given temperature.