Both polarities of the repetitive-sequence, multimeric forms of the satellite RNA of tobacco ringspot virus (sTobRV RNA) cleave autolytically and specifically to generate 359 nucleotide residue (nt), "monomeric" sTobRV RNA. The negative polarity sTobRV (-)RNA also spontaneously ligates to perform the cleaved 3'-to-5'ApG phosphodiester. sTobRV (-)RNA does not conform to the well-studied "hammerhead" structure, which does apply to sTobRV (+)RNA. The cleavage-active sequences of sTobRV (-)RNA are dispersed in two regions of the molecule, forming a natural "ribozyme" system. E, an oligoribonucleotide corresponding to one of these two regions, acted on the other, S, and cleaved it at the ApG phosphodiester. Two segments of base pairing between E and S previously have been identified. We propose a systematic mutagenesis of specific regions of the E-S complex to identify critical nt. The complex also will be subjected to chemical modification to identify tertiary interactions, and a screen has been designed to identify mutations which compensate for inactivating mutations. Novel forms of the self-cleaving sequence have been synthesized, or planned, to facilitate the proposed investigations. We expect that the information gathered will allow us to propose a structural model for the cleavage- ligation-complex. The results will improve our understanding of sTobRV RNA replication and facilitate "ribozyme" design.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
9008172
Program Officer
DeLill Nasser
Project Start
Project End
Budget Start
1990-08-15
Budget End
1994-07-31
Support Year
Fiscal Year
1990
Total Cost
$261,000
Indirect Cost
Name
University of California Davis
Department
Type
DUNS #
City
Davis
State
CA
Country
United States
Zip Code
95618