Multiple cis-acting sites positively regulate Escherichia coli nrd expression

Regulation of nrd expression in Escherichia coli by cis -acting elements was found to be more complex than previously reported. At least five upstream sites appear to positively regulate nrd expression including a Fis binding site, a DnaA binding site, an AT-rich region, an inverted repeat and a 10 bp site between the AT-rich region and the inverted repeat. Double mutants defective in these sites indicate that all sites tested act independently when regulating nrd expression. As the decrease in nrd expression in exponentially growing cultures paralleled the decrease observed in DNA synthesis-inhibited cultures for all single and double mutants, we concluded that nrd is regulated by the same mechanism in these physiological states. As mutants unable to induce nrd expression during inhibition of DNA synthesis also fail to exhibit cell cycle-regulated nrd expression, we conclude that cell cycle nrd regulation is controlled by these same sites. Site-directed mutagenesis was used to show that the absence of an increase in nrd expression during DNA inhibition previously observed for deletion of the AT-rich region results from deletion of both the Fis binding site and the AT-rich region.     

Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli

Expression plasmids encoding random sequence mutant proteins of insulin-like growth factor II (IGFII) were constructed by cassette mutagenesis, to improve the efficiency of IGFII synthesis in Escherichia coli. A pool of oligodeoxyribonucleotide linkers containing random trinucleotide sequences were used to introduce second-codon substitutions into the gene encoding Met-Xaa-Trp-IGFII in expression vectors. E. coli RV308 cells transformed with these vectors synthesized IGFII at levels varying from 0-22% of total cell protein. This variable synthesis is a function of the random second-codon sequence and its corresponding amino acid, Xaa. Our data showed that mRNA stability, protein stability and translational efficiency all contributed to variable expression levels of Met-Xaa-Trp-IGFII in E. coli. Furthermore, an efficiently synthesized IGFII mutant protein, Met-His-Trp-IGFII, was converted to natural sequence IGFII by a simple oxidative cleavage reaction.     

Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2

It has been suggested that Escherichia coli release factor 2 (RF-2) translation is autoregulated. Mature RF-2 protein can terminate its own nascent synthesis at an intragenic, in-phase UGA codon, or alternatively, a +1 frameshift can occur that leads to completion of the RF-2 polypeptide. Translational termination presumably increases with RF-2 concentration, providing negative regulatory feedback. We now show, in lacZ/RF-2 fusions, that translation of a UAG codon at the position of the UGA competes with frameshifting, which proves one postulate of the translational autoregulatory model. We also identify a nearby sequence that is required for high-frequency frameshifting and suggest a constraint for the codon preceding the shift point. Both these sequences are incorporated into a model for frameshifting. Our measurements allow us to compute the relative rates in vivo of these reactions: release factor action, frameshifting and tRNA selection at an amber codon.     

Effects of release factor context at UAA codons in Escherichia coli.

In Escherichia coli, nonsense suppression at UAA codons is governed by the competition between a suppressor tRNA and the translational release factors RF1 and RF2. We have employed plasmids carrying the genes for RF1 and RF2 to measure release factor preference at UAA codons at 13 different sites in the lacI gene. We show here that the activity of RF1 and RF2 varies according to messenger context. RF1 is favored at UAA codons which are efficiently suppressed. RF2 is preferred at poorly suppressed sites.     

Tightly controlled expression systems for the production and purification of Escherichia coli release factor 1

The genes for the protein release factors in Escherichia coli have traditionally proven difficult to maintain on high copy plasmids. We have established here systems which provide for both stable maintenance of the release factor 1 gene on such plasmids, as well as high level overproduction of the release factor 1 protein. The gene is maintained under the control of the inducible trc or tac promoters in the presence of very high levels of lac repressor. A simple and rapid scheme for the purification of RF1 from extracts of cultures carrying these plasmids is also described.     

Rapid and precise mapping of the Escherichia coli release factor genes by two physical approaches.

The termination of protein synthesis in Escherichia coli requires two codon-specific factors termed RF1 and RF2. RF1 mediates UAA- and UAG-directed termination, while RF2 mediates UAA- and UGA-directed termination. The genes encoding these factors have been isolated and sequenced, and RF2 was found to be encoded in two separate reading frames. The map position of RF1 has been reported as 27 min on the E. coli chromosome, while the RF2 map position has not yet been identified. In this study, two new and independent methods for gene mapping, using pulsed field gel electrophoresis and an ordered bacteriophage library spanning the entire chromosome, were used to localize the map position of the RF2 gene. In addition, the location of the RF1 gene was more precisely defined. The RF2 gene is located at 62.3 min on the chromosome, while the RF1 gene is located at 26.7 min. This approach to mapping cloned genes promises to be a rapid and simple means for determining the gene order of the genome.     

Frameshifting by eukaryotic ribosomes during expression of Escherichia coli release factor 2.

Normal translation of the gene for E. coli release factor 2 (RF-2) is characterized by a +1 frameshift event that occurs with 30-50% efficiency. Frameshifting on synthetic RF-2 mRNA by eukaryotic ribosomes has also been observed, even though they lack the capability to interact with the frameshift signal in the same manner as prokaryotic ribosomes. We have mutagenized the sequence of the RF-2 gene to eliminate the need for a frameshift, thereby allowing frameshifting efficiency to be measured by direct comparison of RF-2 production from the mutant with production from the wild-type. Measurements using this approach confirm that frameshifting by rabbit reticulocyte lysate ribosomes occurs at the frameshift region, but with a limited efficiency of approximately 0.4%.     

Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.     

cis-acting sites required for osmoregulation of ompF expression in Escherichia coli K-12.

OmpF and OmpC are major outer membrane proteins which form passive diffusion pores in Escherichia coli K-12. The expression of the structural genes for these proteins, ompF and ompC, is influenced by medium osmotic strength and requires the products of two regulatory genes, ompR and envZ. We have constructed a series of ompF-lacZ fusions containing different regions of ompF to determine sites involved with osmoregulation. These fusions were crossed onto a specialized transducing phage and integrated into the bacterial chromosome in unit copy. By measuring the fluctuations of beta-galactosidase activity in lysogens grown in high versus low osmolarity, we have identified three regions which are necessary. Furthermore, we have determined that, although the OmpR activation site is not sufficient, OmpR is probably essential for ompF osmoregulation.