Comparative analysis of core promoter region: information content from mono and dinucleotide substitution matrices.

We have studied the core promoter region in five sets of promoter sequences by calculating the average mutual information content H (relative entropy). We have used specially constructed substitution matrices to calculate mono and dinucleotide replacements in a given block of aligned sequences. These substitution matrices use log-odds form of scores, which are in bits of information. Here, we constructed and applied nucleotide substitution matrices for the core promoter region to calculate the information content to study the Transcription Start Site (TSS), TATA-box and downstream regions. As expected, the information content decreases with increasing block size. This clearly implies that the TSS region is likely to be 5-10 bases in size (length). We also notice that both in the case of mouse and humans, both TATA-boxes and TSS regions are likely to play important roles in proper transcriptional initiation.

Effect of pH on the structure of rhizopuspepsin.

The crystal structure of rhizopuspepsin has been determined at three different pH values (4.6, 7.0 and 8.0) and compared with the previously reported structure at pH 6.0. A pH-sensitive region in the protein has been identified where certain structural changes take place at pH 8.0. An increase in the mobility of loops, weakening of hydrogen bonding and ionic interactions and a change in the water structure have been observed in this region. The loop between the first and the second beta-strands of the N-terminus shows increased mobility at high pH. This loop is known to be highly flexible in aspartic proteinases, aiding in relocating the N-terminal beta-strand segment in pH-related structural transformations. The observed changes in rhizopuspepsin indicate the triggering of a possible denatured state by high pH. The conformation of the active aspartates and the geometry of the catalytic site exhibit remarkable rigidity in this pH range.

Role of water molecules in the structure and function of aspartic proteinases.

The solvent structure in the crystals of ten aspartic proteinases has been analyzed to find the possible roles of conserved water molecules in their structure and activity. 17 waters have been identified which are common to at least eight of the ten examined enzyme structures. These include the catalytic water molecule, whose direct involvement in the mechanism of action of aspartic proteinases has been proposed previously. There appears to be at least one more functionally important water molecule strategically located to stabilize the flexible 'flap' region during substrate binding. Many other waters stabilize the structure, whilst a few have been found to maintain the active-site geometry required for the function of the enzyme. In particular, two waters related by the approximate molecular dyad are involved in the formation and preservation of a network of hydrogen bonds extending from the active site.