The promoter-proximal, unstable IB region of the atp mRNA of Escherichia coli: an independently degraded region that can act as a destabilizing element.

Differential expression of the genes in the Escherichia coli atp (unc) operon is achieved via control of the translational initiation, translational coupling and mRNA stability of the respective genes. The atpIB region of the polycistronic mRNA is less stable than the remaining seven genes. We have investigated the functional half-lives of the atp genes in reconstructed versions of the operon. In order to be able to do this reliably, we have readdressed the interpretation of the complex functional inactivation data obtained by means of transcriptional inhibition using rifampicin. Our results indicate the usable information to be gleaned from this commonly applied technique, while identifying the potential errors in their quantitative interpretation. We estimate that the functional half-life of atpB is slightly over one-half that of atpE and the other atp genes, while atpI is at least two times less stable than atpB. The instability of the atpI mRNA was also demonstrated by its rapid fragmentation. Relocation of atpIB to a position in the promoter-distal region of the operon between atpG and atpD did not change the inactivation rate of atpB. However, it did destabilize the atpG mRNA. Examination of the physical degradation of atpI mRNA shows particularly rapid cleavage in this gene, thus explaining the destabilization effect. The atpIB segment is therefore an autonomously unstable region that can act as a destabilizing element for upstream-located genes in a polycistronic environment.

Translational regulation of a recombinant operon containing human platelet-derived growth factor (PDGF)-encoding genes in Escherichia coli: genetic titration of the peptide chains of the heterodimer AB.

A new strategy is described for the production of recombinant heteromultimeric proteins using Escherichia coli as host. A recombinant operon was constructed containing modified cDNA sequences encoding the mature A and B chains of human platelet-derived growth factor (PDGF). The relative expression rates of the PDGF genes were varied over a range equivalent to A:B ratios from 0.8 to 3.7 by means of translational regulation. This was achieved using two different translational initiation sequences (TIS) upstream from the respective coding regions, one derived from the E. coli atpE translational initiation region, and the other containing a sequence with extended complementarity to the 3' end of the 16S rRNA. The generation of mature PDGF A and B chains in different relative amounts in E. coli provided the basis for developing a novel procedure for the production of the biologically active PDGF heterodimer AB in large quantities. The general strategy is applicable to the preparation of a wide range of heteromultimeric complexes. Moreover, the described PDGF operon constitutes a compact and versatile model system for studies of the posttranscriptional regulation of gene expression.

Detection and localization of the i protein in Escherichia coli cells using antibodies.

Using antibodies raised against the purified i protein, the expression of the chromosomal uncI gene was demonstrated. The i protein was identified as a component of the cytoplasmic membrane and shown to be present in preparations of Fo or F1Fo. The protein is not associated with the F1 moiety.

Overproduction and purification of the uncI gene product of the ATP synthase of Escherichia coli.

The uncI gene, the first gene of the unc operon, has been cloned into an expression vector carrying the lambda PRPL promoters in tandem orientation and the gene cI857 coding for the thermolabile repressor. Linkage of the uncI gene to an efficient ribosome binding site (the translational initiation region of the uncE gene) resulted in 10-20-fold increased gene expression. The i protein has been extracted from overproducing cells using chloroform/methanol and purified to homogeneity by ion exchange chromatography. Analyzing the products of the uncI gene encoded by different plasmids, we provide evidence that, in contrast to the previously reported data (Walker, J. E., Saraste, M., and Gay, N. J. (1984) Biochim. Biophys. Acta 768, 164-200), the chromosome-encoded i protein contains the N-terminal sequence Ser-Val-Ser-Leu-Val-Ser-Arg and has a molecular weight of 13,504.

The mechanism of translational coupling in Escherichia coli. Higher order structure in the atpHA mRNA acts as a conformational switch regulating the access of de novo initiating ribosomes.

Bacterial genes are commonly transcribed to form polycistronic mRNAs bearing reading frames whose respective translational efficiencies are not independently determined. As in many bacterial operons, expression of the atp genes of Escherichia coli is strongly influenced by translational coupling. The gene pair atpHA is tightly coupled, whereby atpA is translated at least three times more efficiently than atpH. However, there is no fixed stoichiometry of coupling: mutations in atpH lead to increases in the translation ratio (atpA/atpH) of up to approximately 40-fold. We have demonstrated that secondary structure sequestering the atpA translational initiation region (TIR) is important to the coupling mechanism in that it inhibits de novo translational initiation at the atpA start codon. Genetic and structural analyses indicate that this inhibitory structure can be induced to refold into a less inhibitory conformation either by introducing two single-base substitutions or as a result of ribosomes translating atpH. We propose a model in which the secondary structure of the atpA TIR acts analogously to a "gating device" in that it restricts de novo ribosomal initiation until it is "switched" into a more open conformation. This contrasts with the function of a stem-loop structure located immediately downstream of atpA and upstream of the Shine-Dalgarno region of atpG, which was found to inhibit translation, but not to mediate tight coupling. Results obtained using the "specialized" ribosome system of Hui and de Boer ((1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4762-4766) indicate that primarily ribosomes reinitiating after termination on atpH are responsible for inducing refolding of the atpA TIR. The principle of alternative mRNA conformations with different functional properties embodied in the model presented here can only be fulfilled by certain types of structure. It is likely to operate in several steps of prokaryotic gene expression, underlying a range of regulatory events including transcriptional attenuation and translational activation.

Translation of the first gene of the Escherichia coli unc operon. Selection of the start codon and control of initiation efficiency.

The uncI gene is the most poorly expressed gene of the unc operon of Escherichia coli. We examined how the structural features of the translational initiation region (TIR) of this gene relate to the efficiency of its expression. A set of various TIR sequences was created for the uncI gene by coupling synthetic oligodeoxynucleotides to the 5' part of the plasmid-borne gene. Analyses of transcription and translation of the resulting constructs revealed that weak translational initiation efficiency of the uncI TIR sequence is a rate-controlling factor for expression. Moreover, the identification of an endonucleotytic cleavage site within the 5' part of the uncI mRNA by Northern blotting lends further support to the notion that instability of the uncI cistron within the polycistronic unc mRNA represents a further mode of control. The analysis of the roles of the different Shine-Dalgarno regions and putative start codons within the translational initiation region of the uncI gene revealed that the Shine-Dalgarno region III and the GUG codon (codon 4 of the uncI coding sequence described by Walker, J. E., Saraste, M., and Gay, N. J. (1984) Biochim. Biophys. Acta 768, 164-200) define the main site of initiation. However, manipulations of the translational initiation region led to selection of an initiation codon further upstream. The relationship between mRNA sequence and higher order structure on the one hand, and initiation site selection and initiation efficiency on the other, was analyzed.

Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor.

Vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) are members of a dimeric-growth-factor family with angiogenic properties. VEGF is a highly potent and specific mitogen for endothelial cells, playing a vital role in angiogenesis in vivo. The role of PIGF is less clear. We expressed the monomeric splice forms VEGF-165, VEGF-121, PIGF-1 and PIGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system. In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins. The dimers were tested for their ability to promote capillary growth in vivo and stimulate DNA synthesis in cultured human vascular endothelial cells. Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PIGF splice forms, showed mitogenic activity. The results demonstrate that four different heterodimeric growth factors are likely to have as yet uncharacterized functions in vivo.