Craig 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.

Transcription termination in vivo in the leader region of the tryptophan operon of Escherichia coli.

Abstract Internal deletions were isolated which have one terminus in the leader region of the tryptophan ( trp ) operon, the transcribed segment preceding the first major structural gene, and the other terminus in one of the five structural genes. These deletions increase expression of the remaining structural genes of the operon up to ninefold without affecting trp operator function, suggesting, in agreement with the conclusion of Jackson & Yanofsky (1973), that there is a site between the operator and trpE which normally limits operon expression. Quantitative hybridization analyses indicate that in trpR bacteria growing in the presence of excess tryptophan, only one of every eight to ten RNA polymerase molecules which initiate transcription on the operon proceeds beyond the leader region into the structural genes. Consistent with this conclusion is the detection of a major, discrete, trp mRNA species corresponding to the initial 130 to 140 nucleotide pairs of the leader region. In addition, leader sequence oligoribonucleotides are generally present in relative molar excess over those corresponding to structural genes. Since leader deletions do not affect transcription initiation frequency, it appears that there is a site of transcription termination in the leader region of the operon, immediately preceding trpE .

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).

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.

Antitermination of characterized transcriptional terminators by the Escherichia coli rrnG leader region

We have used a plasmid antitermination test system to examine the response of an Escherichia coli rRNA operon antiterminator to transcription through Rho-dependent and Rho-independent terminator-containing fragments. We also monitored transcription through multiple copies of a terminator to explore the mechanism of rrn antitermination. Four principal observations were made about antitermination and transcriptional terminators. (1) The rrn antiterminator mediated efficient transcription through Rho-dependent terminators. (2) Under the influence of the rrn antiterminator, RNA polymerase transcribed through two and three copies of the Rho-dependent 16 S----terminator with nearly the same efficiency as through one. (3) The antiterminator had less effect on fragments containing Rho-independent terminators; the rpoC t fragment and three fragments derived from the rrnB terminator region stopped antiterminated transcription. Four other Rho-independent terminator fragments were weakly antiterminated in our test system. (4) Surprisingly, the strength of these terminator fragments was not strongly related to properties such as the -delta G or number of trailing uridine residues of their canonical Rho-independent structures, but appears to be related to additional downstream terminators. We have drawn the following conclusions from these experiments. First, that ribosomal antitermination primarily reverses Rho-dependent termination by modifying the RNA polymerase elongation complex. Transcription through a 1700 nucleotide, multiple terminator sequence showed that the antiterminator caused persistent changes in the transcription process. Second, that fragments derived from the Rho-independent rrnB and rpoBC terminator regions can effectively stop antiterminated transcription. Third, that efficient in vivo termination may often involve regions with complex multiple terminators.

Nucleotide sequence of the promoter-operator region of the tryptophan operon of Escherichia coli

The nucleotide sequence of the region preceding the transcription initiation site of the tryptophan (trp) operon of Escherichia coli was determined by RNA and DNA sequencing techniques. RNA complementary to this region was synthesized in vivo or in vitro on 80trp transducing phage DNA and isolated by hybridization to the DNA of other transducing phage which contain only the pre-initiation region in common. The RNA was sequenced by analysis of complete RNase A and RNase T1 digestion products and overlapping these oligonucleotides with partial digestion products generated by RNase T1 and carboxymethylated RNase A. DNA sequencing of 5′ end-labelled restriction fragments containing the pre-initiation region was carried out using the hydrazine/dimethyl sulphate procedure of Maxam & Gilbert (1977) . Genetic and biochemical studies indicate that the region analyzed contains the trp promoter and the trp operator. The DNA segment implicated in operator function exhibits twofold rotational symmetry and immediately precedes the transcription start-site. Sequence similarities between the trp promoter region and other promoters are discussed.