SummaryRibosomal RNA is transcribed about twice as fast as messenger RNA in vivo, and this increased transcription rate requires the rrn boxA antitermination system. Because several Nus factors have been implicated in rrn antitermination, we have examined the role of NusB, NusE and NusG in controlling the rate of rrn boxA-mediated transcript elongation. In vivo RNA polymerase transcription rates were determined by measuring the rate of appearance of lacZ transcript using a plasmid that contained an inducible T7 promoter fused to the rrn boxA sequence followed by the lacZ gene. This plasmid was introduced into Escherichia coli mutant strains that can be conditionally depleted of NusG, or that carry a deficient nusB gene or a nusE mutation. We found that, in addition to the rrn boxA antiterminator sequence, both NusG and NusB were required to maintain the high transcription rate. The nusE mutation used in this study may be specific for lambda antitermination, as it did not influence the boxA-mediated increase in transcription rate.

Similarities between lambda and rRNA transcription antitermination have led to suggestions that they involve the same Nus factors. However, direct in vivo confirmation that rRNA antitermination requires all of the lambda Nus factors is lacking. We have therefore analyzed the in vivo role of NusB and NusG in rRNA transcription antitermination and have established that both are essential for it. We used a plasmid test system in which reporter gene mRNA was measured to monitor rRNA antiterminator-dependent bypass of a Rhodependent terminator. A comparison of terminator read-through in a wild-type Escherichia coli strain and that in a nusB::IS10 mutant strain determined the requirement for NusB. In the absence of NusB, antiterminatordependent terminator read-through was not detected, showing that NusB is necessary for rRNA transcription antitermination. The requirement for NusG was determined by comparing rRNA antiterminator-dependent terminator read-through in a strain overexpressing NusG with that in a strain depleted of NusG. In NusGdepleted cells, termination levels were unchanged in the presence or absence of the antiterminator, demonstrating that NusG, like NusB, is necessary for rRNA transcription antitermination. These results imply that NusB and NusG are likely to be part of an RNA-protein complex formed with RNA polymerase during transcription of the rRNA antiterminator sequences that is required for rRNA antiterminator-dependent terminator read-through.All rRNA operons in Escherichia coli have antiterminator sequences in their leader and spacer regions that allow RNA polymerase, modified with protein factors, to transcribe through Rho-dependent terminators of rRNA operons (1, 2, 5, 21). The identities of all of the protein factors have not yet been established, but the RNA sequences required have been determined (5, 37). The rRNA leader region antiterminator features include a region of dyad symmetry, referred to as boxB, and conserved sequences, boxA and boxC (21). The spacer regions do not contain the boxC feature (5). Studies with an entire rRNA operon on a plasmid show that leader region boxA mutations result in a 20 to 25% decrease in the amount of 16S and 23S rRNA (14). Spacer boxA mutations result in an additional 15% decrease of 50S subunits (28). These studies suggest that as a consequence of improper rRNA transcription antitermination (rRNA-AT), there is an increase in Rho-dependent termination that in turn results in a decrease of 16S and 23S rRNA. Mutational studies of the three rRNA antiterminator features, boxB, boxA, and boxC, identified boxA as the essential region for transcription antitermination and boxA alone as sufficient for in vivo readthrough of Rho-dependent terminators (5). However, the other conserved features may have as-yet-unidentified functions in vivo. In addition, the boxA feature by itself stimulates an increase in the RNA polymerase transcription elongation rate from 35 to 65 nucleotides per s on the lacZ gene (38).Although there is no direct evidence, a relationship bet...