Transcripts That Increase the Processivity and Elongation Rate of RNA Polymerase

Abstract

Transcripts encoded by the cis-acting antitermination sites (put sites) of lambdoid phage HK022 promote readthrough of downstream transcription terminators. Proper conformation of the transcripts is essential for activity, since put mutations that prevent the formation of predicted RNA stems prevented antitermination, and suppressor mutations that restore the stems restored antitermination. Antitermination does not appear to require proteins other than RNA polymerase, since put-dependent readthrough of multiple sequential terminators was observed in a purified transcription system consisting of template, polymerase, substrates, and buffer. Transcription of put also increased the elongation rate of polymerase, very likely by suppressing pausing. A mutation that alters the zinc-finger region of the beta' subunit of polymerase specifically prevented the put-dependent increases in terminator readthrough and elongation rate. The simplicity of HK022 antitermination contrasts with that of other known antitermination pathways. We propose that the central effector is a transcript that directly alters the elongation properties of RNA polymerase.

Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules

“…However, the reported nucleoside triphosphate regulation of elongation appears to saturate at ϳ5 M NTPs and thus is unlikely to account for the heterogeneity observed here at 0.5 mM NTPs. Allosteric regulation of E. coli RNAP by specific transcript sequences is well established (19,61,62), but the transcription template used here encodes no known regulatory sequences. On the other hand, the origin of kinetic differences between molecules might be purely conformational; it has been speculated that RNAP can adopt a large number of long-lived conformational substates with differing catalytic properties (23).…”

“…performed on a molecular ensemble) kinetics experiments. These studies (17-19, 21, 22) have been interpreted as supporting complex mechanisms in which individual enzyme molecules can switch between two (or more) distinct, parallel reaction pathways by binding an allosteric regulator (17)(18)(19) or through conformational changes much slower than enzymatic turnover (23). However, uncertainty in the interpretation of macroscopic data can arise if the enzyme preparation contains molecular subpopulations with permanent differences in structure and kinetics.…”

“…A variety of proteins and DNA sequence elements in both prokaryotes and eukaryotes regulate gene expression by altering the extent to which active TECs terminate transcription upstream of or within genes (13-15). The efficiency of termination is strongly dependent on the rate of transcript elongation (16), which is modulated (and termination efficiency thus regulated) by accessory proteins that bind to the TEC as well as by sequence elements in the template or nascent transcript (17)(18)(19)(20). Thus, full understanding of expression regulation requires knowledge of the mechanisms by which the rate of elongation is altered.…”

Balanced branching in transcription termination

“…King et al (58) reported in 1996 that the putL and putR antitermination sequences of the Hong Kong phage HK022 (59), shown in Table 5, caused downstream read-through of a triple terminator consisting of tRЈ from phage followed by the strong E. coli ribosome operon terminators rrn B t 1 and rrn B t 2 . This effect was sensitive to the choice of promoter.…”

Opposite Consequences of Two Transcription Pauses Caused by an Intrinsic Terminator Oligo(U)

“…For example, phage -encoded N protein interacts with Escherichia coli RNA polymerase, exerting an antitermination effect on nutL and nutR terminators of the phage genome (1). In other examples, RNA polymerase interacts with a product transcript rather than an external factor; hence, the antitermination is intrinsic, requiring no external factors, such as the antitermination at the phage HK022 put operon leader (10).…”