Evolutionary comparison of ribosomal operon antitermination function

Abstract

Transcription antitermination in the ribosomal operons of Escherichia coli results in the modification of RNA polymerase by specific proteins, altering its basic properties. For such alterations to occur, signal sequences in rrn operons are required as well as individual interacting proteins. In this study we tested putative rrn transcription antitermination-inducing sequences from five different bacteria for their abilities to function in E. coli. We further examined their response to the lack of one known rrn transcription antitermination protein from E. coli, NusB. We monitored antitermination activity by assessing the ability of RNA polymerase to read through a factor-dependent terminator. We found that, in general, the closer the regulatory sequence matched that of E. coli, the more likely there was to be a successful antitermination-proficient modification of the transcription complex. The rrn leader sequences from Pseudomonas aeruginosa, Bacillus subtilis, and Caulobacter crescentus all provided various levels of, but functionally significant antitermination properties to, RNA polymerase, while those of Mycobacterium tuberculosis and Thermotoga maritima did not. Possible RNA folding structures of presumed antitermination sequences and specific critical bases are discussed in light of our results. An unexpected finding was that when using the Caulobacter crescentus rrn leader sequence, there was little effect on terminator readthrough in the absence of NusB. All other hybrid antitermination system activities required this factor. Possible reasons for this finding are discussed.

FIG. 1.

Plasmids used in the study. Schematic diagrams of plasmids pSL102, pSL103, and pSL115 are shown (29). The genes encoding chloramphenicol acetyltransferase (cat) and β-lactamase (bla) are marked. The blaP and rrnGP2 promoters, the site of the rrn antitermination system insertion (AT), and the fragment containing a Rho-dependent terminator (ter) are also marked (29).

FIG. 2.

Relative chloramphenicol resistance levels of the wild-type and ΔnusB strains containing the AT tester sequences in plasmid pSL103 derivatives (see Materials and Methods for details). P, promoter only (pSL102); P+T, promoter plus terminator (pSL103). All other strains have the indicated AT sequence inserted between the promoter and terminator. Identity of inserted AT sequences: Ec, E. coli; Bs, Bacillus subtilis; Cc, Caulobacter crescentus; Mj, Methanococcus jannaschii; Mt, Mycobacterium tuberculosis; Pa, Pseudomonas aeruginosa; Tm, Thermotoga maritima. Shown are triplicate experiments performed on separate days. The ΔnusB strains have a slower growth phenotype and thus display a lower level of chloramphenicol resistance when measured at the same time as the wild-type strains.

FIG. 3.

Slot blot analysis of expression from the cat and bla genes. See Materials and Methods for experimental details. Duplicate samples were analyzed in this example. Abbreviations are the same as those used in Fig. 2.

FIG. 4.

Alignment of the rrn AT regions of a sample of sequenced Gammaproteobacteria. Residues with more than 50% conservation at each position are shaded. Bars above the sequence denote the extent of the boxA and GT-rich boxC regions. The sequences are taken from published genomic data in the NCBI database.