Recently, we proposed the following model for mRNA degradation in procaryotes. There is no unique target mRNA for its inactivation and initiation of decay. Instead, there is an endoribonuclease that cleaves certain pyrimidine-adenosine (pyr-A) bonds. These targets are protected by associated ribosomes. An initial cleavage is usually near the 5'-end for statistical reasons and the same enzyme """"""""chops off"""""""" large oligonucleotide fragments (perhaps 30 to 200 nucleotides) from the newly-created 5'-end as the last ribosome progresses down the message. The fragments are degraded to mononucleotides by several other RNases. The initial goal of this proposal is to purify the pyr-A enzyme using various chromatographic separations. So far, we have observed its specific activity using 5S rRNA as a substrate. Substrates will be synthesized to define further its specificity as well as other characteristics of its enzymology. Mammalian pancreatic RNase at very low concentrations also has a specificity for pyr-A bonds and the two enzymes will be compared with a view that this activity may be encoded by a family of enzymes distributed throughout Nature. The complete amino acid sequence of the enzyme will be determined. A strain will be constructed in which the synthesis or activity of the RNase can be regulated. Deoxynucleotide probes (15-20 nucleotides) will be synthesized to code for amino acid sequences of the enzyme and used to purify its gene in a lambda library of total E. coli DNA. Alternative procedures will be used, if necessary. The major construction will include exchanging a regulated promoter, e.g., trp, and substituting the gene-containing DNA into the host by transformation and recombination. Strains with temperature-sensitive synthesis or activity of the enzyme will also be constructed. mRNA metabolism will be studied in the new strains and mutations in other RNAse would be added in order to identify all steps in degradation. The 5'- and 3'-ends of gal mRNA of yeast will be identified to see if the same basic mechanism of degradation occurs in a lower eucaryote.
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