Genome-wide transcription start site (TSS) profiles of the enterobacteria Escherichia coli and Klebsiella pneumoniae were experimentally determined through modified 5′ RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5′ UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5′ UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5′ UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization.

For high‐accuracy template‐based‐modeling of CASP7 targets, we have applied a procedure based on the rigorous optimization of score functions at three stages: multiple alignment, chain building, and side‐chain modeling. We applied the conformational space annealing method to a newly developed consistency based score function for multiple alignment. For chain building, we optimized the MODELLER energy function. For side‐chain modeling, we optimized a SCWRL‐like energy function using a rotamer library constructed specifically for a given target sequence. By rigorous optimization, we have achieved significant improvement in backbone as well as side‐chain modeling for TBM and TBM/HA targets. For most TBM/HA targets (17/26), the predicted model was more accurate than the model one can construct from the best template in a posteriori fashion. It appears that the current method can extract relevant information out of multiple templates. Proteins 2007. © 2007 Wiley‐Liss, Inc.

A novel ␤-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the ␤-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The ␤-transaminase showed higher activities toward D-␤-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The ␤-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and ␣-ketoglutarate/ oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of ␤-aminocarboxylic acids in the active site is reversed relative to that of ␣-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the ␤-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic ␣-keto acids such as ␣-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the ␣-carboxylate group of the ␣-amino acids and ␣-keto acids. The ␤-transaminase was used for the asymmetric synthesis of enantiomerically pure ␤-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.

No abstract