The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frameshifting by a mechanism indistinguishable from that of retroviruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.The pol genes of retroviruses are expressed as gag-pol or gag-pro-pol fusion polyproteins (1) formed either by in-frame read-through of termination codons (2, 3) or by ribosomal frameshifting (4-6). Both mechanisms allow for production of multiple proteins from a single, unmodified mRNA.In Rous sarcoma virus (RSV), gag and pol genes overlap, with pol being in the -1 frame with respect to gag (7). In 5% of translations, a -1 frameshifting event allows ribosomes to miss the gag termination codon and continue to translate the pol gene, producing a gag-pol fusion protein (8, 9). A -1 ribosomal frameshifting has also been described in coronaviruses [(+) single-stranded (ss) RNA genomes] (10, 11), phage T7 (12), and in the dnaX gene of Escherichia coli (13)(14)(15). A +1 ribosomal frameshift is seen in the yeast retrotransposon Tyl (16)(17)(18) and in the E. coli release factor 2 (19-21).The signals responsible for -1 ribosomal frameshifting include a "slippery site" heptamer, X XXY YYZ (gag reading frame indicated; X = A, U, or G; Y = A or U; Z = A, U, or C), followed by a stem-loop structure that can be involved in an RNA pseudoknot (4,9,13,20,22,23). A pseudoknot is base pairing of the loop with a sequence 3' of a stem-loop (24, 25). The "simultaneous slippage" model of Jacks et al. (4) proposes that the tRNAs bound at the ribosomal P site to XXY and at the A site to YYZ simultaneously slip back 1 base on the mRNA to pair with XXX and YYY, respectively. Because their nonwobble bases remain properly paired, this can happen at a finite rate (Fig. 1). The stem-loop structure has been demonstrated to be essential for efficient frameshifting in RSV (4), infectious bronchitis virus (23), and the E. coli dnaX gene (13) and is predicted to occur following the slippery site heptamers of a number of other retroviruses (4, 9, 23). RNA secondary structure downstream of the slippery site may slow or stall ribosomes such that they remain in the slippery site longer, thus promoting frameshifting (4).The L-A g...

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex “mitochondrial contact site and cristae organizing system” and its subunits Mic10 to Mic60.

Ribosomal protein S6 of wild-type strains of Escherichia coli contains up to six glutamic acid residues at its C-terminus. The first two residues are encoded by the structural gene for this protein (rpsF) and the rest are added post-translationally. Mutants deficient in this modification were isolated and characterized genetically and biochemically. The S6 protein in these mutants appeared to contain only two glutamic acid residues at the C-terminus as expected. The mutated gene was termed rimK and was mapped at 18.7 min between cmlA and aroA. The rimK gene was cloned into a cosmid vector and its nucleotide sequence determined. Analysis of the transcriptional and translational products of this gene indicates that it encodes a protein with an Mr of 31.5 kDa and that it forms an operon with a gene encoding a 24 kDa protein. An rpsF mutant containing a Glu to Lys replacement in the second residue from the C-terminus of protein S6 was isolated. The S6 protein of this mutant was apparently inaccessible to the RimK modification system. This indicates that the RimK modification system requires the wild-type amino acid sequence at least in the C-terminal region of ribosomal protein S6.

The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope. Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane. The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions. About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes. A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid. This overproduced protein also revealed the dual subcellular localization.The phosphatidyl glycerophosphate (PGP) B phosphatase of Escherichia coli hydrolyzes three substrates: PGP, phosphatidic acid (PA), and lysophosphatidic acid (LPA). Although the membrane localization of PGP phosphatase has not been studied previously, enzymes in the phospholipidbiosynthetic pathway are generally localized in the cytoplasmic membrane in E. coli and Salmonella typhimurium. On the other hand, Bell et al. (2) observed that LPA phosphatase is localized in the outer membrane. Therefore, the finding that the PGP, PA, and LPA phosphatase activities directed by the pgpB gene are not separable genetically (17) raised a question about the substrate specificity and membrane localization of the pgpB gene product. The unusual discovery presented in this paper is that this enzyme is located in both the outer and cytoplasmic membranes. Furthermore, PGP phosphatase activity is higher in the cytoplasmic membrane, whereas PA and LPA phosphatase activities are higher in the outer membrane.To understand this unusual dual subcellular localization, I have cloned this gene and inserted it into an expression vector, a runaway replication plasmid (3) carrying the tac promoter (28), to overproduce this gene product. This study shows that the pgpB gene is a single structural gene coding for an enzyme with all three phosphatase activities. The product of the pgpB gene behaves differently in the cytoplasmic and outer membranes of E. coli. MATERIALS AND METHODSBacterial strains and plasmids. E. coli JM103 (lacJ^) (25) was obtained from New England Biolabs, Beverly, Mass. Strain TI82 (phoA8 pgpB26 recA) was a recA derivative from T174 (17). Other strains are described in Table 1 of the accompanying paper (15).Plasmid pLC26-25 (cysB+ gyrA+ pgpB+) was found among the Clarke and Carbon collection of Col El E. coli hybrid plasmids (6). Plasmids pACYC184, pBR322, and pTI10 were described in the accompanying paper (15). Plasmid pDR540 (28) was from PL Biochemicals, Milwaukee, Wis. Plasmid pMOB45 was described previously (18).An expression vector, pTI5, was constructed by inserting t Present address: Laboratory of Genetics, Department of Biology, The University of Tokyo, Hongo, Tokyo 113, Japa...