Fumiaki Yamao

The ribosomal protein gene cluster of Mycoplasma capricolum

SummaryThe DNA sequence of the part of the Mycoplasma capricolum genome that contains the genes for 20 ribosomal proteins and two other proteins has been determined. The organization of the gene cluster is essentially the same as that in the S10 and spc operons of Escherichia coli. The deduced amino acid sequence of each protein is also well conserved in the two bacteria. The G+C content of the M. capricolum genes is 29%, which is much lower than that of E. coli (51%). The codon usage pattern of M. capricolum is different from that of E. coli and extremely biased to use of A and U(T): about 91% of codons have A or U in the third position. UGA, which is a stop codon in the “universal” code, is used more abundantly than UGG to dictate tryptophan.

Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum

SummaryThe nucleotide sequences of the rrnB 16S ribosomal RNA gene and its 5′-and 3′-flanking regions from Mycoplasma capricolum have been determined. The coding sequence is 1521 base pairs long, being 21 base pairs shorter than that of the Scherichia coli 16S rRNA gene. The 16S rRNA sequence of M. capricolum reveals 74% and 76% identity with that of E. coli and Anacystis nidulans, respectively. The secondary structure model constructed from the M. capricolum 16S rRNA.gene sequence resembles that proposed for E. coli 16S rRNA. A large stem structure can be constructed between the 5′- and 3′-flanking sequences of the 16S rRNA gene. The flanking regions are extremely rich in AT.

Organization of ribosomal RNA genes in Mycoplasma capricolum

SummaryDNA segments carrying rRNA genes of Mycoplasma capricolum have been cloned and characterized by restriction endonuclease mapping, DNA-RNA hybridization and nucleotide sequencing. The M. capricolum genome has two sets of rRNA gene clusters, where the arrangement is in the order of (5′)16S-23S-5S(3′). The spacer region between 16S and 23S rDNA is extremely rich in AT and does not carry any tRNA genes.

Evolutionary dynamics of tryptophan tRNAs in Mycoplasma capricolum.

SummaryMycoplasma capricolum uses two tryptophan codons, the “universal” nonsense condon UGA and the universal codon UGG. The bacterium contains two tryptophan tRNAs, one with anticodon UCA, (U: 2′-O-methyl U derivative), and the other with CCA (5′-C: partially 2′-O-methylated). tRNAUCA would translate codons UGA and probably UGG by wobbling. tRNACCA is much less charged by tryptophan in the cells than tRNAUCA, and the intracellular amount of tRNACCA is 5–10 times lower than that of tRNAUCA. The genes for these two tRNAs are separated by a terminator-like structure in a single operon. In vitro transcription experiments suggest that the predominance of tRNAUCA over tRNACCA results from the attenuation of transcription by this terminator-like structure.

The Genome of Mycoplasma capricolum

Publisher Summary This chapter discusses the genome of Mycoplasma capricolum. The simplest way to estimate the approximate number of genes encoded in the genome is to count the number of the gene products in the cell. The entire protein content of M. capricolum was analyzed by two-dimensional polyacrylamide gel electrophoresis of O’Farrell. Acidic proteins were separated using isoelectrofocusing gels for the first dimension with a gradient of pH 5-7 and basic proteins with a gradient of pH 7-9. A total of about 350 spots, 200 in the acidic and 150 in the basic range, was detected. Under the same conditions, the numbers of the whole cell proteins of E. coli and B. subtilis are about 1,100. The results indicate that at least 350 genes for proteins are expressed in the growing M. capricolum cells. The average molecular weight of proteins is about 40,000. The number of expressed genes would represent roughly the number of genes for proteins encoded in the genome. The small number of the protein spots is consistent with the view that mycoplasmas contain only a limited number of the genes necessary for growth and that most, if not all, of them must be expressed in the growing cells.

Genetics of Nonsense Suppressor tRNAs in Escherichia coli

The genetic approach to tRNA originated in the study of nonsense suppressors in Escherichia coli that could specifically restore nonsense mutations in bacteria or bacteriophages. Three codons, UAG (amber), UAA (ocher), and UGA (opal), do not code for an amino acid due to the lack of tRNAs with anticodons corresponding to these codons, called nonsense codons. They are not normally present within the coding regions of genes, but can be generated by nonsense mutations, resulting in the termination of peptide chain elongation at the site. Accordingly, if a mutation occurs in a tRNA gene that alters the coding specificity to recognize a nonsense codon, the altered tRNA may suppress the nonsense mutations in other genes by inserting a specific amino acid during the process of translation. Although the suppression may take place in various ways (for reviews, see Gorini 1971; Steege and Soll 1979), nonsense suppression has been generally attributed to the tRNA suppression. Missense or frameshift mutations are also suppressed by altered tRNAs specific to each mutation (for a review, see Steege and Soll 1979). In the case of suppressor gene mutations, a mutant normally shows positive character ( su + ) differing from ordinary genes and is dominant over its allelic wild-type gene ( su − ). Accordingly, the detection of strains carrying nonsense suppressors or the isolation of new suppressor mutants from su − strains is relatively easy in genetic operation, and, in fact, many different nonsense suppressors (e.g., su + 1 , su + 2, su + B, su + C , etc.) have been detected before they were fully understood. They can...

Molecular cloning of ribosomal protein genes from MYcoplasma capricolum

SummaryA BglII-fragment from the Mycoplasma, capricolum DNA cloned into pBR322 has been found to contain a cluster of ribosomal protein genes. The recombinant plasmid, pMCB1088, includes a 9 kilobase-pair insert that codes for at least eight ribosomal proteins of M. capricolum. The protein genes are expressed in Escherichia coli cells.