Evidence that Rho and NusA are involved in termination in the rplL-rpoB intercistronic region

Abstract

The frequency of transcription of the ribosomal protein and RNA polymerase gene segments of the rpJKAJL-rpoBC gene cluster was measured for Escherichia coli K-12 strains carrying mutations in the genes for transcriptional termi,nation factors. The results of our study suggest that Rho increases and that both NusA and the product of sfrB decrease termination frequency in the rplL-rpoB intercistronic region.The concentration of RNA polymerase core enzyme in growing Escherichia coli is determined by the rate of synthesis of the P and ' polypeptides (8,15). The genes that encode these polymerase subunits (rpoBC) are the distal elements in' a gene cluster that also contains four ribosomal protein genes (rplKAJL) (1,10,14,17,18,23,27,31,32). Two major promoters, rplKp and rplJp, are present in this gene cluster ( Fig. 1) (14,27,32). Hience, the rpoBC genes are cotranscribed with at least the rplJL genes. Moreover, it has been reported that many transcripts initiated at rplKp do not terminate after rplA, but continue through rplJp, thus suggesting that all six genes may be cotranscribed (6).Even though the RNA polymerase subunit genes are cotranscribed with upstream ribosomal protein genes, their frequency of transcription is about 20% of that of the upstream genes (7). 'This difference is accounted for by a transcriptional attenuator (atn) in the rplL-rpoB intercistronic region which terminates approximately 80% of the transcripts during steady-state growth (2, 27). DNA sequence analysis has suggested that there is a sequence characteristic of a transcriptional terminator at 43 to 73 base pairs (bp) beyond the end of rplL (22,25). The proposal that this indeed functions as a terminator was further strengthened by Si nuclease and RNA sequence analyses of an in vivo transcript whose 3' end mapped 69 bp beyond rplL (3). The RNA transcript from this region can form an 11-bp stem followed by five consecutive uridine residues (see Fig. 3). This is the canonical structure of a simple or factorindependent terminator. However,'20% of the transcripts continue through this sequence, and various experiments have suggested that the termination frequency may be altered under certain conditions (5,19,20). These results suggest that the function of this sequence may be more complicated than that of a simple constitutive terminator.A number of factors in addition to the RNA polymerase core enzyme and the RNA sequence have been demonstrated to participate in transcriptional termination. The best studied of these is the Rho protein (28). A general class of terminators, referred to as Rho-dependent terminators, has been recognized (13,29), but no simple consensus sequence is evident. What appear to be required are both a nucleotide * Corresponding author. sequence which causes RNA polymerase to pause and an adjacent stretch of RNA devoid of secondary structures (29). Other factors involved in regulating termination are the products of the nusA, nusB, and nusE genes. These were first identified as host genes necessary for N-mediated antit...

Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism

“…These data are consistent with a role for RfaH in enhanced elongation of transcription. RfaH also increases steady-state mRNA levels from several other gene clusters in E. coli and S. typhimurium (4,5,(26)(27)(28).…”

“…RfaH also enhances steady-state levels of mRNA of several other loci, including a plasmid-borne hlyCABD operon (2), the traY-Z operon on the F plasmid (4), the rplK-rpoC gene cluster (27), and the rfaQ-K gene cluster (26,28), as well as the homologous rfa operon in Salmonella typhimurium (5). Although the precise mechanism of action of RfaH is unknown, all of the available data support a model for RfaH involvement in enhanced transcriptional elongation.…”

Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12

“…Creeger et al (6) have presented evidence that sfrB affects the levels of both galactosyl transferase and glucosyltransferase II in extracts of E. coli. A broader role for sfrB as an antiterminator is suggested by the observation of Ralling and Linn (14) that the product of sfrB decreases termination frequency in the rplL-rpoB intercistronic region.…”

Transcription-frequency-dependent modulation of an attenuator in a ribosomal protein-RNA polymerase operon requires an upstream site

“…We have previously found evidence, both in vivo (Ralling & Linn, 1987) and in vitro (Linn & Greenblatt, 1992), that NusA decreases termination frequency at rpoBa. To test whether NusA played a role in the transcription-frequency-dependent modulation of rpoBa, two pairs of lacZ transcriptional fusions were used to assay rpoBu readthrough in wild-type cells and three nusA mutants, nusAIO(Cs) (Schauer et al, 1987), nusAl (Friedman, 1971) and nusAl1 (Ts) (Nakamura et al, 1986).…”

“…Previously, we observed that the level of transcription of rpoB relative to the upstream rpZJL genes was decreased in a nusAl mutant strain (Ralling & Linn, 1987). Subsequently, we found that the addition of either NusA or NusG to a purified in vitro transcription system increased the readthrough of rpoBa (Linn & Greenblatt, 1992).…”