; R o o t E n t r y F 5 @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l m 5 m 5 3 4 5 6 7 8 9 : ; < F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; * S M h } # E " < | Q h j d z Q 3 : rWk ? }< 1 ; V R P 7 G s iD l 9507046 Weiner The CCA-adding enzyme synthesizes and regenerates the 3 terminal CCA of tRNA. It is not yet known, from either a biochemical or an evolutionary perspective, whether the CCA-adding enzyme is a specialized polymerase or a sequence specific terminal transferase. The project will test the hypothesis that the enzyme may resemble a polymerase in structure, with amino acid residues functioning as an internal template for ribonucleotide addition. These experiments plan an integrated approach to the structure, function, and evolution of this unusual enzyme. Specifically, PCR will be used to obtain additional sequences for phylogenetically diverse CCA adding enzymes, and these sequences will be compared to identify conserved regions and residues, as well as phylogenetically variable domains of the enzyme; and the effect of mutations in conserved regions and residues, as well as the effect of deleting phylogenetically variable domains, will be tested both in vitro and in vivo, to identify regions critical for function. %%% The CCA adding enzyme is the only known polymerase that can synthesiz e a defined nucleotide sequence without using a nucleic acid template. It is not yet known--from either a biochemical or an evolutionary perspective--whether the CCA adding enzyme is a specialized polymerase or a sequence specific terminal transferase. The P.I. proposes an integrated approach to the structure, function, and evolution of the CCA adding enzyme. He will obtain sequences for phylogenetically diverse CCA adding enzymes and compare the protein sequences of these diverse CCA adding enzymes to identify conserved regions as well as phylogenetically variable domains. The P.I. hypothesizes that the CCA adding enzyme may be structurally or functionally related to the family of template dependent polymerases, and this proposal will also test that hypothesis. If so, previous studies of the enzyme can now be revisited in light of the newly emerging view of polymerase structure and function. *** @ ....()()))()() ; Oh +' 0 $ H l D h R:WWUSERTEMPLATENORMAL.DOT marcia steinberg marcia steinberg @ M 5 @ S u m m a r y I n f o r m a t i o n ( 2 @ m 5 @ T Õ Microsoft Word 6.0 2 e = e " " " " " " " L L L L L d n L C x | | | | | | | # f W T 4 " | | | | | | " " | x | | | | " | " | 6 > " " " " | | 5 | 9507046 Weiner The CCA-adding enzyme synthesizes and regenerates the 3 terminal CCA of tRNA. It is not yet known, from either a biochemical or an evolutionary perspective, whether the CCA-adding enzyme is a specialized polymerase or a sequence specific terminal transferase. The project will test the hypothesis that the enzyme may resemble a polymerase in structure, with amino acid residues functioning as an internal template for ribonucleotide addition. These experiments plan an integrated approach to the structure, function, and evolution of this unusual enzyme.
