Glucose kinase has a regulatory role in carbon catabolite repression in Streptomyces coelicolor.

A glucose kinase (glkA) mutant of Streptomyces coelicolor A3(2) M145 was selected by the ability to grow in the presence of the nonmetabolizable glucose analog 2-deoxyglucose. In this glkA mutant, carbon catabolite repression of glycerol kinase and agarase was relieved on several carbon sources tested, even though most of these carbon sources are not metabolized via glucose kinase. This suggests that catabolite repression is not regulated by the flux through glucose kinase and that the protein itself has a regulatory role in carbon catabolite repression. A 10-fold overproduction of glucose kinase also results in relief of catabolite repression, suggesting that excess glucose kinase can titrate the repressing signal away. This could be achieved directly by competition of excess glucose kinase with its repressing form for binding sites on DNA promoter regions or indirectly by competition for binding of another regulatory protein.

Structural analysis of a group II intron by chemical modifications and minimal energy calculations.

Folding of the yeast mitochondrial group II intron aI5c has been analysed by chemical modification of the in vitro synthesised RNA with dimethylsulfate and diethylpyrocarbonate. Computer calculations of the intron secondary structure through minimization of free energy were also performed in order to study thermodynamic properties of the intron and to relate these to data obtained from chemical modification. Comparison of the two sets of data with the current phylogenetic model structure of the intron aI5 reveals close agreement, thus lending strong support for the existence of a typical group II intron core structure comprising six neighbouring stem-loop domains. Local discrepancies between the experimental data and the model structures have been analyzed by reference to thermodynamic properties of the structure. This shows that use of the latest refined set of free energy values improves the structure calculation significantly.

Structure-function relationships in a self-splicing group II intron: a large part of domain II of the mitochondrial intron aI5 is not essential for self-splicing.

An oligonucleotide-directed deletion of 156 nucleotides has been introduced into the yeast mitochondrial group II intron al5 (887 nt). The deletion comprises almost all of domain II, which is one of the six phylogenetically conserved structural elements of group II introns. This mutant displays reduced self-splicing activity, but results of chemical probing with dimethylsulphate suggest that sequences at the site of the deletion interfere with the normal folding of the intron. This is supported by computer analyses, which predict a number of alternative structures involving conserved intron sequences. Splicing activity could be restored by insertion of a 10-nucleotide palindromic sequence into the unique Smal site of the deletion mutant, resulting in the formation of a small stable stem-loop element at the position of domain II. These results provide a direct correlation between folding of the RNA and its activity. We conclude that at least a large part of domain II of the group II intron al5 is not required for self-splicing activity. This deletion mutant with a length of 731 nucleotides represents the smallest self-splicing group II intron so far known.

Mitochondrial biogenesis: recent developments and insights.

Biosynthesis of a functional mitochondrion requires the coordinate expression of genes in both mitochondrial and nuclear DNAs. In yeast, three mitochondrial genes are split and RNA splicing plays a pivotal role in their expression. The recent finding that some introns are capable of self-splicing activity in vitro has permitted analysis of the mechanisms involved in RNA catalysis and may eventually shed light on the evolution of splicing mechanisms in general. Most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and imported by the organelle. The availability of cloned genes coding for several constituent subunits of the ubiquinol-cytochrome c reductase, which are imported by mitochondria, has allowed study of selected steps in the addressing of proteins to mitochondria and their intercompartmental sorting within the organelle. Recent developments are discussed.

Mutations at the lariat acceptor site allow self-splicing of a group II intron without lariat formation.

The fifth intron in the gene for cytochrome c oxidase subunit I in yeast mitochondrial DNA is of the group II type and is capable of self-splicing in vitro. The reaction results in lariat formation, concomitant with exon-exon ligation and does not require a guanosine nucleotide for its initiation. It is generally assumed, but not formally proven, that the first step in splicing is a nucleophilic attack of the 2'-hydroxyl of the branchpoint nucleotide (A) on the 5'-exon-intron junction. To investigate the role of intron sequences in recognition of the 5'-splice junction and the ensuing event of cleavage and lariat formation, mutations have been introduced at and around the branchsite. Results obtained show that although branchpoint attack and subsequent lariat formation are strongly preferred events under conditions normally used for self-splicing, addition of a single T residue at intron position 856, a mutation which brings the branchpoint adenosine into a basepair, leads to a conditionally active intron, which at high ionic strength catalyses exon-exon ligation in the absence of lariat formation. Comparable behaviour is also observed with the branchpoint A deletion mutant. The implications of these findings for the mechanism of self-splicing of group II introns are discussed.