IF3-mediated suppression of a GUA initiation codon mutation in the recJ gene of Escherichia coli.

A mutational change of the initiation codon to GUA was found to reduce, but not abolish, expression of the recJ gene of Escherichia coli. Specific mutations in translational initiation factor IF3 have been isolated as second-site suppressors of this GUA initiation codon mutation. One of these, infC135, with an arginine-to-proline change at amino acid 131, completely restores a wild-type phenotype to recJ GUA initiation codon mutants and acts in a semidominant fashion. The infC135 mutation increased expression of RecJ from the GUA mutant but had no effect on the normal GUG start. The infC135 mutation also abolished autoregulation of IF3 in cis and in trans. The behavior of this IF3 mutant suggests that it has specifically lost its ability to abort initiation from poor initiation codons such as GUA of recJ and the AUU of infC. Because of the impact of IF3 on recJ, a recombination and repair gene, this role of IF3 must be general and not restricted to translation genes. The dominance of infC135 suggests that the other functions of IF3, for instance its ability to bind to 30S ribosomes, must remain intact. Although the ability to discriminate among initiation codons has been lost in the infC135 mutant, translational initiation was still restricted to the normal initiation site in recJ, even in the presence of a closely juxtaposed alternative initiation codon. Because the recJ gene lacks a canonical Shine-Dalgarno sequence, other unknown features of the mRNA must serve to specify the initiation site.     

Expression of the Escherichia coli pcnB gene is translationally limited using an inefficient start codon: a second chromosomal example of translation initiated at AUU

Expression of the gene pcnB, encoding the dispensable Escherichia coli poly(A) polymerase (PAPI), which is toxic when overproduced, was investigated. Its promoter was identified and found to be moderately strong when used to express a beta-galactosidase reporter. Expression levels were not affected by increasing or decreasing PcnB concentration. Translation of pcnB was found to initiate from the non-canonical initiation codon AUU. The only other coli gene reported to use AUU as initiation codon is infC, which encodes the initiation factor IF-3. AUU, in common with other rarely used initiation codons, is discriminated against by IF-3, resulting in the aborting of most AUU-promoted initiation events. This enables AUU to form part of an autoregulatory circuit controlling IF-3 production. We show that InfC discrimination reduces PcnB production fivefold. This is the first instance of this mechanism being used to limit severely the production of a potentially toxic product.     

The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo.

The amino acid sequence of the Dsg protein is 50% identical to that of translation initiation factor IF3 of Escherichia coli, the product of its infC gene. Anti-E. coli IF3 antibodies cross-react with the Dsg protein. Tn5 insertion mutations in dsg are lethal. When ample nutrients are available, however, certain dsg point mutant strains grow at the same rate as wild-type cells. Under the starvation conditions that induce fruiting body development, these dsg mutants begin to aggregate but fail to develop further. The level of Dsg antigen, as a fraction of total cell protein, does not change detectably during growth and development, as expected for a factor essential for protein synthesis. The amount of IF3 protein in E. coli is known to be autoregulated at the translational level. This autoregulation is lost in an E. coli infC362 missense mutant. The dsg+ gene from Myxococcus xanthus restores normal autoregulation to the infC362 mutant strain. Dsg is distinguished from IF3 of E. coli, other enteric bacteria, and Bacillus stearothermophilus by having a C-terminal tail of 66 amino acids. Partial and complete deletion of this tail showed that it is needed for certain vegetative and developmental functions but not for viability.     

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.     

Sequence of a 1.26-kb DNA fragment containing the structural gene for E.coli initiation factor IF3: presence of an AUU initiator codon

The nucleotide sequence of a 1.26-kb pair DNA fragment containing the structural gene for Escherichia coli initiation factor IF3 has been determined. An open reading frame of 540 nucleotides is found at the position predicted by genetic studies. The amino-acid sequence deduced from the DNA sequence accounts for a molecular weight 20 530. The important feature of the coding DNA sequence is the presence of AUU as the translational initiator codon. It is 11 bases downstream of the center of a GGAGG sequence, which can strongly pair with the sequence CCUCC near the 3' terminus of 16S rRNA. The primary DNA sequence in the region of the AUU initiator codon and its role in compensating a reduced codon-anticodon interaction in initiation complex formation are discussed.     

Non-canonical translation mechanisms in plants: efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence.

The 5' untranslated leader (Omega sequence) of tobacco mosaic virus (TMV) genomic RNA was utilized as a translational enhancer sequence in expression of the 17 kDa putative movement protein (pr17) of potato leaf roll luteovirus (PLRV). In vitro translation of RNAs transcribed from appropriate chimeric constructs, as well as their expression in transgenic potato plants, resulted in the expected wild-type pr17 protein, as well as in larger translational products recognized by pr17-specific antisera. Mutational analyses revealed that the extra proteins were translated by non-canonical initiation at AUU codons present in the wild-type Omega sequence. In the plant system translation initiated predominantly at the AUU codon at positions 63-65 of the Omega sequence. Additional AUU codons in a different reading frame of the Omega sequence also showed the capacity for efficient translation initiation in vitro. These results extend the previously noted activity of the TMV 5' leader sequence in ribosome binding and translation enhancement in that the TMV translation enhancer can mediate non-canonical translation initiation in vitro and in vivo.     

同义密码子用语的位置依赖

研究了在大肠杆菌编码区不同位置上的同底密码子用语,发现许多氨基酸的密码子用语在转译起始区有显著的变化,仅有少数氨基酸在转译区有较弱的变化,由于密码子用语与基因表达关系密切。这些结果与实验发现的编码区5‘端密码子用对表达的重要性是一致的。更进一步的结果还暗示了哪些密码子在特定位置的使用可能会影响基因表达。     

Polyhedrin initiator codon altered to AUU yields unexpected fusion protein from a baculovirus vector

A recombinant baculovirus expression vector was constructed to express the core (capsid) protein of the hepatitis B virus. Along with the expected 21-kDa polypeptide, a second 24-kDa protein was observed. Immunoprecipitation and immunoblotting using a rabbit polyclonal anticore antiserum demonstrated that the two proteins were related. The core gene originally was cloned in-frame with the polyhedrin initiator codon that had been altered to AUU as a means of preventing fusion protein formation. A transient expression assay revealed expression of the 24-kDa protein was prevented if a frame-shift mutation was created upstream of the HBV core translation start site. These results suggest that the 24-kDa protein was the result of an unexpectedly high level of translation initiation at the AUU codon that gave rise to a polyhedrin-HBV core fusion protein. The 24-kDa core protein was shown to be a polyhedrin fusion protein by immunoblotting with an antipolyhedrin antiserum, and initiation at the AUU was demonstrated by amino terminal protein sequencing. Methods to prevent undesired fusion protein expression using this or similar vectors are discussed.