Catherine L. Squires

Antitermination of E. coli rRNA transcription is caused by a control region segment containing lambda nut-like sequences

We have localized the antitermination system involved in E. coli ribosomal RNA transcription and compared it with antitermination in the lamboid bacteriophages. In vivo experiments with gene-fusion plasmids were used to examine the ability of specific areas of the rrnG control region to convert an ordinary transcription complex into antitermination transcription complex. A 67 bp restriction fragment immediately following the rrnG P2 promoter decreased transcription termination about 50%. This fragment contains box A-, box B-, and box C-like sequences similar to those in lambda nut loci. It also caused transcripts from lac and hybrid trp-lac promoters to read through a transcription terminator. Translation through the 67 bp segment or reversal of its orientation resulted in complete loss of antitermination activity. We conclude that the E. coli ribosomal RNA operons possess an antitermination system similar to that used by the bacteriophage lambda.

Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli.

We have compared the expression of the seven ribosomal RNA operons (rrn) of Escherichia coli and their responses to a variety of physiological and genetic perturbations. We used a set of rrn promoter fusion constructs in their native chromosomal positions to examine effects of chromosomal location on rrn operon expression and the same set of fusions on lambda lysogens to assay intrinsic promoter strengths independent of chromosome context. In its native chromosomal location, expression of the rrnH operon was significantly lower than expected. This effect was not attributable to weak promoter activity and was dependent on the growth medium. The rrnE operon had reduced promoter activity relative to the other ribosomal operons in minimal medium and thus appears to have abnormal growth rate regulation. The ribosomal RNA operons showed varied responses to amino acid starvation; expression of rrnD was inhibited most. There was only a slight increase in rrn transcription in response to a temperature shift (30 degrees C to 42 degrees C) and the differences between individual operons was very small. The rrnG operon showed a significantly lower response than the other ribosomal RNA operons to a depletion of the rrn transcription activator, Fis, and thus appears to have decreased Fis‐mediated transactivation. Finally, the chromosomal fusion strains were used to study the effect on growth rate of inactivating each rrn operon. In fast growth conditions, loss of certain rrn operons caused subtle decreases in growth rate on complex medium.

Ribosomal RNA operon anti-termination: Function of leader and spacer region box B-box A sequences and their conservation in diverse micro-organisms

All Escherichia coli rrn operons show a common motif in which anti-terminator box B-box A sequences occur twice, first in the leader and again in the 16 S-23 S spacer. In this study we have analyzed several aspects of rrn anti-termination by leader and spacer anti-terminator sequences. Using DNA synthesis and a plasmid test system, we incorporated random changes into the leader anti-terminator region and examined these mutations for their ability to read through a strong terminator. We also examined anti-termination by synthetic box A and by rrn spacer region sequences. Information derived from these experiments was used to search the rrn sequences of other micro-organisms for possible anti-termination features. Our principal conclusions were that: (1) box A was sufficient for terminator readthrough; (2) we could show no positive requirement for box B in our test system; (3) many of the negative anti-terminator mutations caused a promoter up-effect in the absence of a terminator; (4) the search of rrn operons from other micro-organisms revealed that anti-terminator-like box B-box A sequences exist in leader and spacer regions of both eubacteria and archaebacteria. The frequent occurrence of this pattern suggested that the E. coli rrn anti-termination motif is widespread in nature and has been conserved in microbial evolution.

Depletion of functional ribosomal RNA operons in Escherichia coli causes increased expression of the remaining intact copies.

The synthesis of ribosomal RNA is a complex and highly regulated process. To study this process, we have used deletion‐insertions to disrupt sequentially from one to four of the seven rRNA (rrn) operons on the Escherichia coli genome. Inactivation of four rrn operons caused a 2.3‐fold increase in the expression of a chloramphenicol acetyl transferase reporter gene fused to the tandem promoters of rrnA and a similar increase in the expression of the trp tRNA gene at the end of rrnC. This reflected enhanced expression of the remaining operons to compensate for having only three intact copies. The elevated expression was caused by an increase in both transcription initiation and RNA polymerase elongation rates specifically on rrn operons and occurred in the absence of changes in the intracellular concentration of ppGpp, suggesting that ppGpp is not involved in the regulation of this phenomenon. We discuss these results in relation to the ribosome feedback inhibition model described by Nomura and coworkers.

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three‐dimensional cryo‐electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912–910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885–887 (GGG) and 888–890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A‐site tRNA‐binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.

Nucleotide sequence of the 5' end of tryptophan messenger RNA of Escherichia coli.

Abstract The 5′-terminal sequence of the tryptophan (trp) operon messenger RNA of Escherichia coli was determined by a combination of in vivo and in vitro 32P-labeling techniques. A leader sequence of approximately 166 nucleotides precedes the start codon for the polypeptide specified by the operator-proximal structural gene, trpE. The leader sequence contains an AUG codon (positions 27 to 29) in a region which binds ribosomes (Platt et al., 1976) and is followed in phase by the tandem chain termination codons, U-A-A-U-G-A (positions 120 to 125). The inferred DNA sequence has a region of high G + C content (positions 126 to 137) followed by a region of high A + T content (positions 138 to 156). Studies in vivo (Bertrand et al., 1976) and in vitro (Lee et al., 1976) suggest that transcription termination occurs in the vicinity of these G + C and A + T-rich regions and that termination may be regulated (Bertrand & Yanofsky, 1976).

Interaction of the trp repressor and RNA polymerase with the trp operon

Abstract An in vitro transcription system for the tryptophan operon of Escherichia coli was used to determine the mechanism by which trp † repressor inhibits transcription of the operon. It was found that trp repressor-binding prevents binding of RNA polymerase to the operon. It was also observed that when polymerase is prebound to the trp promoter, repressor cannot prevent it from transcribing. These results suggest the existence of a functional overlap between trp promoter and trp operator regions and indicate that repressor acts by preventing RNA polymerase attachment to the promoter region of the operon.

Antiterminator‐dependent modulation of transcription elongation rates by NusB and NusG

Ribosomal 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.

Termination of transcription in vitro in the escherichia coli tryptophan operon leader region

Abstract Transcription of the wild-type tryptophan (trp) operon of Escherichia coli was examined in vitro. Virtually all RNA polymerase molecules which initiate transcription at the trp promoter cease transcription within the leader region of the operon after synthesizing about 145 nucleotides of leader RNA, and thus rarely transcribe the structural genes of the operon. Transcription stops with approximately equal frequency at either of two adjacent nucleotide pairs within an A + T-rich region, giving rise to transcripts with U-rich 3′ termini. The site of transcription termination is in a segment of the leader region proposed on the basis of genetic and biochemical evidence to contain a new regulatory element, a transcription attenuator, which functions in controlling the maximum level of expression of the operon.

In Vivo Effect of NusB and NusG on rRNA Transcription Antitermination

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 Rho-dependent 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, antiterminator-dependent 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 NusG-depleted 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.