High-level expression of genes under control of cro translation initiation signals in Escherichia coli

The expression of several genes in Escherichia coli under the control of the lambda p_R promoter and translation initiation signals of the lambda cro gene were studied. Fusions were made in frame at the initiation codon and/or with 5′ translated cro fragments. Expression fluctuated strongly when genes were fused directly at the ATG, whereas constructs, which encode hybrid genes that include at least the first nine codons of the cro gene, always directed high-level synthesis. These fusion proteins were mainly intracellularly precipitated. Our results confirm the poor reliability of ATG vectors for the expression of cloned genes. On the other hand, useful levels of expression are obtained when genes are fused to 5′ cro coding sequences, presumably due to an efficient ribosome binding site configuration.     

Identification of an Intragenic Ribosome Binding Site That Affects Expression of the uncB Gene of the Escherichia coli Proton-Translocating ATPase (unc) Operon

The uncB gene codes for the a subunit of the F_o proton channel sector of the Escherichia coli F₁ F_o ATPase. Control of expression of uncB appears to be exerted at some step after translational initiation. Sequence analysis by the perceptron matrices (G. D. Stormo, T. D. Schneider, L. Gold, and A. Ehrenfeucht, Nucleic Acids Res. 10:2997–3011, 1982) identified a potential ribosome binding site within the uncB reading frame preceding a five-codon reading frame which is shifted one base relative to the uncB reading frame. Elimination of this binding site by mutagenesis resulted in a four- to fivefold increase in expression of an uncB′-′lacZ fusion gene containing most of uncB. Primer extension inhibition (toeprint) analysis to measure ribosome binding demonstrated that ribosomes could form an initiation complex at this alternative start site. Two fusions of lacZ to the alternative reading frame demonstrated that this site is recognized by ribosomes in vivo. The results suggest that expression of uncB is reduced by translational frameshifting and/or a translational false start at this site within the uncB reading frame.     

The SOS response is induced by replication fork blockage at a Ter site located on a pUC-derived plasmid: dependence on the distance between ori and Ter sites

A new model system for the study of the SOS response has been developed. In this system the response is induced by blocking the replication fork at a Ter site located in pUC-derived plasmids. Blockage of the fork is dependent on the expression of the Ter binding protein, Tus, encoded on another plasmid, in which the tus gene is under the control of the ara promoter. SOS induction can, therefore, be controlled by arabinose. The extent of the SOS response was monitored by measuring the activity of β-galactosidase, expressed from a lacZ gene fused to the 5′ region of the sfiA gene, a typical SOS-responsive gene. Expression of the fusion gene is completely dependent on recA ⁺ and lexA ⁺ genes. Using this system, we found that the distance between the ori and Ter sites is directly correlated with the strength of SOS induction. The properties of this system are discussed.     

A new mutation inEscherichia coli K12,isfA,which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.     

Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.

Two forms of initiation factor 2, (IF-2α, M_r, 118,000 and IF-2β, M_r 90,000) have been isolated from Escherichia coli extracts and tested for their ability to support β-galactosidase synthesis in a phage DNA-directed in vitro protein synthesis system. Although both forms are equally active in supporting the binding of fMet-tRNA to ribosomes only IF-2α functions in β-galactosidase synthesis.     

Mechanism of regulating the expression of λN gene by ribosomal protein at translational level

In ribosomal protein S12 mutant or L24 mutant the expression of λN gene was depressed at translational level. To study its mechanism the λN gene region of λN -lacZ gene fusion was trimmed from its 5′ end to 3′ end with DNA exonuclease III (DNA cxoIII) in order to alter the TIR (translational initiation region) and the ding region of λN gene. After DNA sequencing 23 species of different λN-lacZ fused genes were obtained. The β-galactosidase activities of these deletants in ribosomal protein mutant were compared with that in wild type strain. The result indicated that (i) S12 mutant could affect 305 subunit’s binding to the TIR of λN gene messenger and cause the difficulty in forming 30s initiation complex and then decrease the efficiency of translational initiation; (ii) in S12 mutant the coding region of λN gene alw affected the expression λN gene; (iii) in L24 mutant the inhibition of λN gene expression was not related to translational initiation and the 5′ end of the coding region of λN gene, but related to the 3′ end of λN gene.     

Location of ribosome binding sites on the right end of lambda DNA.

The locations of ribosome binding sites on the right end of bacteriophage lambda DNA were determined by measurement of the positions of ribosomes bound to single-stranded DNA visualized by electron microscopy. A total of ten ribosome binding sites were found between map co-ordinates 0.90 and 1.0. Four of these ribosome binding sites probably correspond to the polypeptide initiation sites for genes Q (0.910), S (0.928), R (0.936) and Rz (0.945). Six other ribosome binding sites were found which presumably indicate the presence of new lambda genes. Four of these ribosome binding sites (0.958, 0.967, 0.975, 0.995) are located to the right of Rz, which is the most rightward known lambda gene. A ribosome binding site is located at 0.923, between genes Q and S, in or near the 6 S RNA sequence. Another is located left of gene Q at 0.900.     

Structure analysis of three T7 late mRNA ribosome binding sites

The 5′-terminal regions of the three T7 late RNA species IIIb, IV and V have been characterized. These regions contain the protein synthesis initiation sites for the T7 genes 17, 9 and 10, respectively. Each of these is located between 60 and 90 nucleotides from the 5′ terminus of an in vitro synthesized RNA species. The sequence 5′ A-C-U-U-U-A-A-G-Pu-A-G-Pu, which is common to these ribosome binding regions, contains an impressive stretch of complementarity to the sequence 5′ A-C-C-U-C-C-U-U-A, at the 3′ terminus of 16 S ribosomal RNA. The nuclease mapping technique of Wurst et al. (1978) has been used to probe intramolecular structural interactions involving these initiation regions in the RNA. My results indicate that all three initiation codons, together with other portions of the ribosome binding regions are protected, under non-denaturing conditions, against the actions of both the single-strand-specific nuclease S₁ and RNAase T₁.