From Genome to Life:

VITRESHCHAK Alexey

Institute for Problems Information Transmission RAS Bolshoj Karetnyj, Moskow 101447, Russia

title: Computer analysis of regulation of genes, encoding aminoacyl-tRNA synthetases and amino acid biosynthetic proteins in Gram positive bacteria: T-box RNA regulatory element. Prediction of regulation of new genes, including amino acid transporters.

Introduction. Computer comparative analysis is a powerful method of prediction of the RNA secondary structure. It has been used for prediction of both regulatory and structural RNAs. A somewhat different approach is to predict gene regulation by analysis of RNA patterns. We have used it to analyze the T-box regulatory elements in Gram positive bacteria. It is experimentally known a number of T-box elements in Bacilllus subtilis, Bacillus. stearothermophilus, Lactococcus lactis and Staphylococcus aureus. The set of known T-boxes from these bacteria contains 14 genes and operons, encoding 11 aminoacid tRNA synthetases (ileS, tyrZ, tyrS, trpS, hisS, valS, pheST, thrS, thrZ, leuS, argS) and 3 aminoacid biosynthetic operons (cysE-cysS, ilv-leu, trp). The T-box regulatory element consists of the alternative RNA secondary structures (the terminator and antiterminator conformations) and the conservative sequences boxes (T-box, F-box, AG-box, GNTG-box, AGTA-box and aminoacid anti-anticodon box). Uncharged aminoacid-tRNA is the inducer of transcription. It binds to the RNA structure (with T-box and anti-anticodon site) and promotes formation of the antiterminator. Results. Using known T-boxes we constructed a common pattern and scaned the available genomes using the RNApattern program, developed for this purpose. It used the set of parameters: length of helixes and loops, distance between helixes and location of conservative nucleotides. The common pattern includes antiterminator and terminator conformation and 14-bp conservative box (T-box). New T-boxes were found in the genomes of Bacillls. subtilis, Bacillus anthracis, Bacillus stearothermophilus, Bacilllus halodurans, Staphylococcus aureus, Enterococcus faecalis, Lactococcus lactis, Clostridium acetobutilicum, Clostridium difficile, Streptococcus pneumoniae, Streptococcus mutans, Streptococcus pyogenes, Streptococcus equi and Deinococcus. radioduras. In B. subtilis new T-boxes were observed upstream of aminoacyl-tRNA synthetase genes, genes encoding enzymes and genes of unknown function. Comparison of sets of T-box-regulated genes in the analysed genomes shows, that while the majority of these genes is still constituted by aminoacyl-tRNA synthetase genes, the fraction of enzyme and transporter genes is much larger than previously thought. The regulation by T-boxes does not seem particulary conservative: in a number of cases only one or two genes in a set of ortologs is regulated by this mechanism. The trp operon, regulated in B. subtilis by binding the TRAP protein to a periodic sequence in the leader mRNA region, is regulated by T-boxes in B. anthracis, S. aureus, C. acetobutilicum, S. pneumoniae and S. mutans, wheras in L. lactis it is under dual regulation by TRAP and T-box. Several genes (hom, trp, yheL, yocR) have double T-boxes in the upstream regions. The reason for this is not clear. On the other hand, sometimes regulation by T-boxes survives changes in the operon structure. Thus, the 7-gene operon ilvBNC-leuABCD is regulated by a single T-box in B. subtilis, whereas in Clostridium spp. there are additional T-boxes between the ilv and leu genes. More detailed analysis of the metabolic map will be done in order to understand the functional implications of this arrangement. Overall, this study represents the first attempt to perform a large-scale analysis of RNA regulatory pattern and we believe that even the preliminary results presented here are very encouraging. This study was partially supported by grants from INTAS (99-1476) and HAMI (55000309).

This is joint work with A.A.Mironov and M.S.Gelfand.