RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli

Efficient expression in Escherichia coli (E. coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E. coli codon usage, both fused to the E. coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region. High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG. A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1%. These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting. They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation. In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site. That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses. mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region.     

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.     

Initiation of translation with Pseudomonas aeruginosa phage PP7 RNA: nucleotide sequence of the coat cistron ribosome binding site.

Initiation complex formation between PP7 RNA and ribosomes of Pseudomonas aeruginosa and Escherichia coli has been investigated. The PP7 RNA fragments protected by both species of ribosome have been isolated, and their sequences have been determined. Only one binding sites is available on the intact PP7 RNA strand, and this site is recognized by ribosomes of both species. The PP7 RNA binding site is approximately 38 nucleotides long. It contains two AUG sequences and a purine-rich segment near the 5'-end that is complementary to segments near the 3'-ends of the 16S ribosomal RNA's of both P. aeruginosa and E. coli. In order to establish which of the AUG codons acts as the initiator, the H2N-terminal amino acid sequence of PP7 coat protein was determined. This sequence is compatible with the codon sequence following the second AUG codon. The extent of the reaction of PP7 RNA with E. coli ribosomes is greater than with P. aeruginosa ribosomes, but our results do not indicate a qualitative difference in the initial interaction between intact PP7 RNA and the ribosomes of either species.     

Translation of chloroplast-encoded mRNA: potential initiation and termination signals.

A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively. As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22. A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes. In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation. A SD sequence could not be located in 6% of the chloroplast sequences. We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation. We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E. coli.     

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.     

Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.     

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.     

Emerging links between initiation of translation and human diseases

Some diseases are caused by mutations that perturb the initiation step of translation by changing the context around the AUG(START) codon or introducing upstream AUG codons. The scanning mechanism provides a framework for understanding the effects of these and other structural changes in mRNAs derived from oncogenes, tumor suppressor genes, and other key regulatory genes. In mRNAs from mutated as well as normal genes, translation sometimes initiates from an internal AUG codon. Sanctioned mechanisms that allow this, including leaky scanning and reinitiation, are discussed. Thrombopoietin mRNA is an example in which translation normally initiates from an internal position via an inefficient reinitiation mechanism. Mutations that restructure this mRNA in ways that elevate production of thrombopoietin cause hereditary thrombocythemia, demonstrating that some mRNAs are designed deliberately with upstream AUG codons to preclude efficient translation and thus to prevent harmful overproduction of potent proteins. While upstream AUG codons in certain mRNAs thus play an important regulatory role, the frequency of upstream AUG codons tends to be exaggerated when cDNA sequences are compiled and analyzed. Because the discovery of mutations that perturb translation usually begins with cDNA analysis, some misunderstandings vis-a-vis the interpretation of cDNA sequences are discussed.     

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.     

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.     

A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs.

The 5'-non-translated regions (5'NTR) of human immunoglobulin heavy chain binding protein (BiP), Antennapedia (Antp) ofDrosophilaand human fibroblast growth factor 2 (FGF-2) mRNAs are reported to mediate translation initiation by an internal ribosome binding mechanism. In this study, we investigate predicted features of the higher order structures folded in these 5'NTR sequences. Statistical analyses of RNA folding detected a 92 nt unusual folding region (UFR) from 129 to 220, close to the initiator AUG in the BiP mRNA. Details of the structural analyses show that the UFR forms a Y-type stem-loop structure with an additional stem-loop in the 3'-end resembling the common structure core found in the internal ribosome entry site (IRES) elements of picornavirus. The Y-type structural motif is also conserved among a number of divergent BiP mRNAs. We also find two RNA elements in the 5'-leader sequence of human FGF-2. The first RNA element (96 nt) is 2 nt upstream of the first CUG start codon located in the reported IRES element of human FGF-2. The second (107 nt) is immediately upstream of the authentic initiator AUG of the main open reading frame. Intriguingly, the folded RNA structural motif in the two RNA elements is conserved in other members of FGF family and shares the same structural features as that found in the 5'NTR of divergent BiP mRNAs. We suggest that the common RNA structural motif conserved in the diverse BiP and FGF-2 mRNAs has a general function in the internal ribosome binding mechanism of cellular mRNAs.     

AUG is the only initiation codon in eukaryotes

An analysis of mutants of the yeast Saccharomyces cerevisiae indicates that AUG is the sole codon capable of initiating translation of iso-1-cytochrome c. This result with yeast and the sequence results of numerous eukaryotic genes indicate that AUG is the only initiation codon in eukaryotes; in contrast, results with Escherichia coli and bacteriophages indicate that both AUG and GUG are initiation codons in prokaryotes. The difference can be explained by the lack of the t6 A hypermodified nucleoside (N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]threonine) in prokaryotic initiator tRNA and its presence in eukaryotic initiator tRNA.     

mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae

The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1-Δ disruption, which stabilized mRNAs that had premature termination codons, resulting in wild-type levels. The following major conclusions were reached by measuring β-galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide-long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position ?3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A>G>C>U. Upstream out-of-frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (?3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.