TTG serves as an initiation codon for the ribosomal protein MvaS7 from the archaeon Methanococcus vannielii

Abstract

The ribosomal protein MvaS7 from the methanogenic archaeon Methanococcus vannielii is a protein of 188 amino acids, i.e., it is 42 amino acids longer than previously suggested. The triplet TTG serves as a start codon. The methanogenic translation initiation region that includes the rare TTG start codon is recognized in Escherichia coli.The gene for ribosomal protein MvaS7 from the methanogenic archaeon Methanococcus vannielii is part of a transcriptional unit which corresponds to the eubacterial streptomycin operon. Apart from the presence of two additional open reading frames and the gene for MvaS10, the organization of the streptomycin operon of M. vannielii (open reading frame 1 [ORF1]-ORF2-MvaS12-MvaS7-EF2-EF1␣-MvaS10) is identical to that of Escherichia coli. In E. coli the S10 gene is the first gene in the S10 operon. On the basis of the nucleotide sequence, MvaS7 was described as a protein of 146 amino acids (16). More recent homology studies of ribosomal S7 proteins from the archaea Haloarcula marismortui, Halobacterium morrhuae, and M. vannielii suggested that MvaS7 should be more than 40 amino acids longer than previously suggested (14).Analysis of the nucleotide sequence preceding the ATG start codon proposed by Lechner et al. (16) revealed an amino acid sequence which shows a high degree of homology to the sequences of the other archaeal S7 proteins. Two triplets, an ATT and a TTG, which are known as unusual start codons in eubacteria (9) and archaea (18), are located in frame upstream from the originally proposed ATG start codon.In this communication we report that MvaS7 is a protein of 188 amino acids, i.e., 42 amino acids longer than originally proposed. Furthermore, we demonstrate that TTG is used as a start codon and that the methanogenic translation initiation region containing TTG as a start codon is recognized in E. coli.Cloning and expression in E. coli of three size variants of the gene encoding MvaS7. Three codons, namely, ATT (Fig. 1, position 2172), TTG (Fig. 1, position 2190), and ATG (Fig. 1, position 2317), are potential candidates for the start codon of the MvaS7 gene. To express the three possible MvaS7 variants in E. coli BL21(DE3) (21), we have chosen vector pET 11a (6), since the expression of cloned genes can be strictly regulated. If MvaS7, the homolog of the autoregulatory protein S7 of the streptomycin operon in E. coli (4), functioned in E. coli, we could not exclude the possibility that the basal expression level of MvaS7 in the uninduced state might affect growth of the E. coli host. To clone the three MvaS7 gene variants, we have used the oligonucleotides shown in Fig. 1 as primers to make PCR products containing an NdeI site and a BamHI site. The PCR protocol and cloning of the PCR products into pET 11a via pUC 18 are detailed in reference 10. To allow cloning and efficient expression, the rare start codons ATT and TTG were replaced by an ATG. The constructs were designated pMvaS7 Identification of MvaS7. Our aim was to identify MvaS7 in the two-dimensional protein patt...

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