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.Transcription antitermination is a ubiquitous mechanism used in the regulation of gene expression in bacteriophages, bacteria, viruses, and possibly also archaea and eukaryotes (12,22). Antitermination circumvents termination events, leading to increased expression of the genes for which it is used. In addition to regulation by other sophisticated mechanisms, transcription of the seven rRNA operons of Escherichia coli is also regulated by transcription antitermination (29, 41). Efficient and balanced synthesis of the three rRNA species, 16S, 23S, and 5S, depends on an antitermination mechanism that modifies the transcription elongation complex into a form that efficiently expresses the entire operon (24,38,39). The antitermination event is signaled by an RNA element, the antitermination site (AT site), consisting of sequences designated boxB, boxA, and boxC. Antitermination requires the association of proteins NusA, NusB, and NusG, as well as ribosomal proteins, with the transcription elongation complex (28,37,46,47). Both the proteins and the AT site reveal sequence homologies across even distantly related species of bacteria (4). For example, examination of rrn leader and spacer regions from widely differing bacteria in the general location of the E. coli AT sequences reveals that AT features are readily recognized on the basis of sequence elements alone. Thus, although only E. coli and Mycobacterium tuberculosis AT sites have been...

Transcription rate and transcript length drive formation of chromosomal interaction domain boundaries

“…This mutation strongly reduced the intensity of the domain boundary at the van locus; however, transcription of this mutant rsaA (ATG→TAA) was severely diminished, likely due to Rho‐dependent transcription termination (Figs and EV3B–D). Thus, to retain high levels of transcription while preventing translation, we inserted a Caulobacter anti‐termination sequence (Arnvig et al , ) into the leader sequence of rsaA . RT–PCR analysis confirmed that rsaA transcription in this strain (anti‐termination + ATG→TAA) was restored to near wild‐type levels (Fig EV3B–D), and S‐layer protein extraction confirmed the absence of rsaA translation (Fig EV3E).…”

Transcription, processing and function of CRISPR cassettes in Escherichia coli

“…As CRISPR transcripts are not translated, it is likely that special antitermination mechanisms exist to ensure efficient transcription of CRISPR RNA. In the case of rDNA transcription, a poorly characterized antitermination system that relies on an upstream rut element ensures efficient production of full‐sized transcripts (Arnvig et al ., 2008). No sequences matching the rut sequence are present in CRISPR transcripts.…”

A multiplex CRISPR interference tool for virulence gene interrogation in an intracellular pathogen

“…While the addition of the strong Ptet promoter did enhance gene silencing by distal spacers ( Figure 2B), gene silencing polarity within the MC arrays was still not fully overcome ( Figure 3), indicating that additional factors likely influenced crRNA production. BoxA elements are often found upstream of long non-coding RNAs, like the 16S RNA (35), where they are bound by the Nus complex to prevent Rho-dependent transcription termination (36)(37)(38). Stringer et.…”