Abstract
In early stage Xenopus oocytes, transcription factor IIIA (TFIIIA) and 5S rRNA form a 7S RNP particle, which is used to store the RNA in cytoplasm. Starting at stage III, TFIIIA is replaced by ribosomal protein L5 on the RNA. Despite using different recognition elements, association of TFIIIA and L5 to 5S rRNA is mutually exclusive due to the overlapping binding sites. It is of importance to determine the topology of TFIIIA and L5 on 5S rRNA in order to understand the mechanism behind the exchange of 5S rRNA between these two proteins. To pinpoint the chemical groups on 5S rRNA which are important for the binding of TFIIIA and L5, nucleotide analog interference mapping (NAIM) was used. A variety of adenosine and guanosine analogs were chosen, which contain modifications to the base, ribose or phosphate moieties. TFIIA interference sites are clustered in loop A and loop E, which is consistent with a recent crystal structure. However, some features have been identified which are not apparent in that structure. For example, strong interference has been observed with phosphorothioate adenosine at position 13, which can not be accounted for by the crystal structure. Removal of the 2¡¦ hydroxyl group at A11 causes pronounced interference, which can be reversed by replacement with fluorine; yet there is no hydrogen bond to this moiety in the crystal structure. Interference sites for L5 are located in helix III and loop C. The interference by m£`A£S and 7dA£S analogs at A50 and the DAP£S analog at position A49 indicate that the two unpaired adenosines in helix III form an A-platform upon binding L5. The m£`A£S analog at A11 enhances the binding of L5, which indicates that the protein likely binds to loop A by stacking on A11. Interestingly, TFIIIA also binds to loop A through a stacking interaction between Trp 177 and A11. This is the only example where these two structurally distinct proteins make similar contacts to the 5S rRNA.
A 30 kDa TFIIIA fragment is observed in the late stage oocytes when the TFIIIA-L5 exchange reaction occurs. An in vitro exchange experiment shows that a 30 kDa peptide corresponding to the N-terminal portion of TFIIIA can be replaced by L5 on 5S rRNA, while the full-length TFIIIA can not be replaced, indicating that programmed cleavage of TFIIIA at the C-terminal end controls the exchange reaction.
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