Use of a portable ribosome-binding site for maximizing expression of a eukaryotic gene in Escherichia coli

To maximize expression of a eukaryotic gene in Escherichia coli, a series of plasmids were constructed containing various synthetic ribosome-binding sites (RBS). These sites consist of a Shine-Dalgarno (SD) region (with translation stop codons in all three reading frames) positioned at distances 5-9 nucleotides (nt) from the AUG initiator codon of the gene coding for human T-cell growth factor (TCGF or IL-2). The region encompassing the RBS through the TCGF structural gene from each of these plasmids was inserted as a 'cassette' into seven different E. coli expression vectors, and TCGF production was measured. Our results demonstrate a greater than 2000-fold range of TCGF synthesis dependent upon the promoter and the synthetic RBS used. The translational efficiency of the TCGF gene was found to be influenced by the quality of the RBS, which is in part determined by the external sequence context of this site. The synthetic RBS, containing the necessary information for the translation initiation process, readily accessible by restriction sites, should be of general usefulness in obtaining maximum expression of eukaryotic genes in E. coli.     

Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region.

A unique genetic selection was devised at the HIS4 locus to address the mechanism of translation initiation in Saccharomyces cerevisiae and to probe sequence requirements at the normal translational initiator region that might participate in ribosomal recognition of the AUG start codon. The first AUG codon at the 5' end of the HIS4 message serves as the start site for translation, and the -3 and +4 nucleotide positions flanking this AUG (AXXAUGG) correspond to a eucaryotic consensus start region. Despite this similarity, direct selection for mutations that reduce or abolish ribosomal recognition of this region does not provide any insight into the functional nature of flanking nucleotides. The only mutations identified that affected recognition of this region were alterations in the AUG start codon. Among 150 spontaneous isolates, 26 were shown to contain mutations in the AUG start codon, including all +1 changes (CUG, GUG, and UUG), all +3 changes (AUA, AUC, and AUU), and one +2 change (ACG). These seven mutations of the AUG start codon, as well as AAG and AGG constructed in vitro, were assayed for their ability to support HIS4 expression. No codon other than AUG is physiologically relevant to translation initiation at HIS4 as determined by growth tests and quantitated in his4-lacZ fusion strains. These data and analysis of other his4 alleles are consistent with a mechanism of initiation at HIS4 as proposed in the scanning model whereby the first AUG codon nearest the 5' end of the message serves as the start site for translation and points to the AUG codon in S. cerevisiae as an important component for ribosomal recognition of the initiator region.     

Translation of the F protein of hepatitis C virus is initiated at a non-AUG codon in a +1 reading frame relative to the polyprotein

The hepatitis C virus (HCV) genome contains an internal ribosome entry site (IRES) followed by a large open reading frame coding for a polyprotein that is cleaved into 10 proteins. An additional HCV protein, the F protein, was recently suggested to result from a +1 frameshift by a minority of ribosomes that initiated translation at the HCV AUG initiator codon of the polyprotein. In the present study, we reassessed the mechanism accounting for the synthesis of the F protein by measuring the expression in cultured cells of a luciferase reporter gene with an insertion encompassing the IRES plus the beginning of the HCV-coding region preceding the luciferase-coding sequence. The insertion was such that luciferase expression was either in the +1 reading frame relative to the HCV AUG initiator codon, mimicking the expression of the F protein, or in-frame with this AUG, mimicking the expression of the polyprotein. Introduction of a stop codon at various positions in-frame with the AUG initiator codon and substitution of this AUG with UAC inhibited luciferase expression in the 0 reading frame but not in the +1 reading frame, ruling out that the synthesis of the F protein results from a +1 frameshift. Introduction of a stop codon at various positions in the +1 reading frame identified the codon overlapping codon 26 of the polyprotein in the +1 reading frame as the translation start site for the F protein. This codon 26(+1) is either GUG or GCG in the viral variants. Expression of the F protein strongly increased when codon 26(+1) was replaced with AUG, or when its context was mutated into an optimal Kozak context, but was severely decreased in the presence of low concentrations of edeine. These observations are consistent with a Met-tRNA_i-dependent initiation of translation at a non-AUG codon for the synthesis of the F protein.     

Effectiveness of distal gene translation in polycistrons depends upon the arrangement of regulatory signals on a template

The role of the translational terminator and initiator signals arrangement for two adjacent genes in polycistronic mRNA has been studied. Semisynthetic beta-galactosidase gene (lacZ) of E. coli and fragment of phage M13 DNA (with promoter PVIII, gene IX, and part of gene VIII) were used for constructing of the IX-VIII-lacZ artificial polycistronic operon. Cloning of the constructs into pBR322 vector resulted in a number of pLZ381N plasmids differing by the mutual arrangement of gene VIII translation terminator codon and SD site and initiator codon (SD-ATG-region) of lacZ gene. The mutual arrangement of gene VIII terminator codon and SDlacZ-ATG region has been altered by means of deletions and insertions that have not affected lacZ translation initiation signals. The beta-galactosidase (beta-Gal) synthesis in E. coli harbouring different types of pLZ381N plasmids has been found to depend on type of cistron coupling (gene VIII and lacZ). The overlapping of terminator and initiator codons (ATGA) for genes VIII and lacZ (type I of polycistrons) provide approximately equal translational level for both cistrons. On the other side, levels of beta-Gal synthesis in case of polycistrons type II (gene VIII stop-codon position at the beginning of SDlacZ or 10 nucleotides upstream) were 20-30 times as high as for type I. Differences in beta-Gal levels have also been found for variants of VIII-lacZ coupling in types IV and III polycistrons (the SDlacZ-ATG region in 27-50 nucleotides downstream from the proximal cistron VIII stop-codon, which, in turn, is 41 nucleotides upstream this terminator). These data cannot be explained on the basis of possible secondary structure including the SDlacZ-ATG region and other parts of polycistronic mRNA. In all these cases similarly stable stem-loop structures have been found. Therefore, the arrangement of the translation termination and initiation signals for two adjacent genes in essential for distal gene translation efficiency. One can imagine that ribosome or its 30S subpartical, stalling on the proximal gene terminator codon, affects the distal gene translation initiation.     

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

We determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine–Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5'-AGGA-3' to 5'-UUUU-3' results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon–anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon–anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.     

Influence of the three nucleotides upstream of the initiation codon on expression of the Escherichia coli lacZ gene in Saccharomyces cerevisiae.

By introducing synthetic oligonucleotides into a lacZ-yeast expression vector a set of 47 plasmids (out of 64 possible) was generated, differing only in the three bases immediately upstream of the AUG initiation codon of the Escherichia coli lacZ gene. Expression of the beta-galactosidase fusion protein encoded by the different plasmids was determined in Saccharomyces cerevisiae by immunogel electrophoresis. Among the clones tested we found a factor 3 difference in expression. A slight nucleotide preference was found in positions -3(A > G > C = U) and -2 (G > C = U > A). The choice of the nucleotide at position -1 immediately 5' of the AUG did not effect translation efficiency. Increasing homology to the yeast consensus sequence (AAAAAAAUGUCU) was not concomitant with an increased translation efficiency. Our results indicate that the choice of nucleotides immediately preceding the initiation codon in yeast does not dramatically influence translation efficiency, as in prokaryotes or higher eukaryotes.     

MicroReview Control of translation initiation in Saccharomyces cerevisiae

The first observations regarding the control of translation initiation in the yeast Saccharomyces cerevisiae were made by Fred Sherman and his colleagues in 1971. Elegant genetic studies of the CYC1 gene resulted in the formulation of 'Sherman's Rules' for translation initiation as follows: (i) AUG is the only initiator codon. (ii) the most proximal AUG from the 5' end of a message will serve as the start site of translation; and (iii) if the upstream AUG codon is mutated then initiation begins at the next available AUG in the message. Hidden within these rules is the mechanism of eukaryotic translation initiation, as these very same rules were later shown to apply to higher eukaryotic organisms and were formulated into the scanning model. However, only in the past five years has yeast been taken seriously as an organism for studying the mechanism of eukaryotic translation initiation. The basis for this is that the yeast genes for at least four mammalian translation initiation factor homologues have been identified and the number is growing. Similar factors suggest similar mechanisms for translation initiation between yeast and mammals. For some translation initiation factors, the genetics of yeast has provided new insights into their function. A mechanism for regulating translation initiation in mammalian cells is now evident in yeast. It seems clear that the molecular genetics of yeast coupled with the available in vitro translation system will provide a wealth of information in the future regarding translational control and regulatory mechanisms. The purpose of this review is to summarize what is known about translational control in S. cerevisiae.     

Mutant analysis of Prevotella sp. plaA-lacZ fusion protein expression in Escherichia coli: support for an essential role of the stem-loop.

This study investigated the involvement of RNA folding in the synthesis of a fusion protein with beta-galactosidase activity. The coding gap region of the Prevotella loescheii adhesin gene plaA was fused in-frame with the Escherichia coli lacZ gene on plasmid pSK105. N-Terminal sequencing of the expressed plaA-lacZ protein indicated that it resulted from translational initiation at a fortuitous ribosomal-binding site within the plaA sequence at nt 570. Specific mutations were introduced in the stem-loop region that precedes the gap sequence. Analysis of stem-loop mutants, together with the introduction of compensatory mutations that restored activity, supports a requirement for stem-loop formation within the plaA sequence preceding the translational initiation site. A mutation reducing the predicted size of the loop, but preserving the stem structure, inactivated fusion protein synthesis. A suppressor mutation predicted to restore the size of the loop restored efficient fusion protein synthesis. In addition, the sequence preceding the translational start site of the plaA-lacZ fusion has several similarities to sequences that function as translational enhancers in prokaryotes. These include a stem-loop structure, an A-U rich region preceding the initiation codon, and a region of homology to 16S rRNA.     

Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli.

In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.     

Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes

The cDNA sequences encoding mature human interleukin 2 (IL2) and beta-interferon (INF beta), respectively, were fused with various translational initiation regions and inserted into two different types of expression vector. The relative levels of expression of the two genes and the functional stability of their respective mRNAs were examined in vivo in Escherichia coli hosts. The addition of the 30-bp sequence, found immediately upstream of the E. coli atpE gene Shine-Dalgarno (SD) sequence, to the translational initiation regions of IL2 and INF beta increased the expression of both these genes by a factor of 6-10. Thus this sequence, which naturally acts within the E. coli atp operon to enhance the translational initiation frequency of the atpE gene, can increase the expression of other genes in E. coli. It may exemplify a specific type of recognition signal for the E. coli translational apparatus.     

Genetic Characterization of the Saccharomyces Cerevisiae Translational Initiation Suppressors Sui1, Sui2 and Sui3 and Their Effects on His4 Expression

Saccharomyces cerevisiae strains containing mutations of the HIS4 translation initiation AUG codon were studied by reversion analysis in an attempt to identify components of the translation initiation complex that might participate in initiation site selection during the scanning process. The genetic characterization of these revertants identified three unlinked suppressor loci: SUI1, SUI2 and sui3, which when mutated restored the expression of the HIS4 allele despite the absence of the AUG initiator codon. Both sui1 and sui2 are recessive and cause temperature-sensitive growth on enriched medium. The temperature-sensitive phenotype and the ability to restore HIS4 expression associated with either sui1 or sui2 mutations cosegregate in crosses. SUI3 mutations are dominant and do not alter the thermal profile for growth. None of the mutations at the three loci suppresses known frameshift, missense or nonsense mutations. Each is capable of suppressing the nine different point mutations of the initiator codon at HIS4 or HIS4-lacZ as well as a two base change (ACC) and a three base deletion of the AUG codon, suggesting that the site of suppression resides outside the normal initiator region. sui1 and sui2 suppressor mutations were mapped to chromosomes XIV and X, respectively. Suppression by sui1, sui2 and SUI3 mutations results in 14-, 11- and 47-fold increases, respectively, relative to isogenic parent strains, in the expression of a HIS4 allele lacking the initiator AUG codon. Part of this increase in the HIS4 expression by sui2 and SUI3 can be attributed to increases of HIS4 mRNA levels, presumably mediated by perturbation of the general amino acid control system of yeast.