1. The effects of variations in substrates on the kinetic properties of Escherichia coli RNase H were studied using antisense oligonucleotides of various types hybridized to complementary oligoribonucleotides. The enzyme displayed minimal sequence preference, initiated cleavage through an endonucleolytic mechanism near the 3' terminus of the RNA in a DNA-RNA chimera and then was processively exonucleolytic. Phosphorothioate oligodeoxynucleotides hybridized to RNA supported cleavage more effectively than phosphodiester oligodeoxynucleotides. Oligonucleotides comprised of 2'-methoxy-, 2'-fluoro- or 2'-propoxy-nucleosides did not support RNase H1 activity. 2. The Km and Vmax. of cleavage of RNA duplexes with full phosphorothioate oligodeoxynucleotides were compared with methoxy-deoxy 'gapmers', i.e.; oligonucleotides with 2'-methoxy wings surrounding a deoxynucleotide centre. Such structural modifications resulted in substantial increases in affinity, but significant reductions in cleavage efficiency. The initial rates of cleavage increased as the deoxynucleotide gap size was increased. Multiple deoxynucleotide gaps increased the Vmax. but had little effect on Km. 3. The effects of several base modifications on the site of initial cleavage, processivity and initial rate of cleavage were also studied.

No abstract

No abstract

No abstract

In order to circumvent limitations of sequence based methods in the process of making functional predictions for proteins, we have developed a methodology that uses a sequence-to-structure-to-function paradigm. First, an approximate three-dimensional structure is predicted. Then, a threedimensional descriptor of the functional site, termed a Fuzzy Functional Form, or FFF, is used to screen the structure for the presence of the functional site of interest (Fetrow et al., 1998; Fetrow and Skolnick, 1998). Previously, a disulfide oxidoreductase FFF was developed and applied to predicted structures obtained from a small structural database. Here, using a substantially larger structural database, we expand the analysis of the disulfide oxidoreductase FFF to the B. subtilis genome. To ascertain the performance of the FFF, its results are compared to those obtained using both the sequence alignment method BLAST and three local sequence motif databases: PRINTS, Prosite, and Blocks. The FFF method is then compared in detail to Blocks and it is shown that the FFF is more flexible and sensitive in finding a specific function in a set of unknown proteins. In addition, the estimated false positive rate of function prediction is significantly lower using the FFF structural motif, rather than the standard sequence motif methods. We also present a second FFF and describe a specific example of the results of its whole-genome application to D. melanogaster using a newer threading algorithm. Our results from all of these studies indicate that the addition of three-dimensional structural information adds significant value in the prediction of biochemical function of genomic sequences.