Mathias Brännvall

The residue immediately upstream of the RNase P cleavage site is a positive determinant

We have studied the importance of the residue at the position immediately upstream of the RNase P RNA cleavage site using model substrates that mimic the structure at and near the cleavage site of the tRNA(His) precursor. The various model substrates were studied with respect to cleavage site recognition as well as the kinetics of cleavage using M1 RNA, the catalytic subunit of Escherichia coli RNase P. Our studies showed that the identity of the residue immediately upstream of the cleavage site critically influences both these aspects. Among the ones tested, U is the preferred nucleotide at this position. Hence, these findings rationalize why most bacterial tRNA(His) genes/transcripts harbor a U immediately upstream of the RNase P cleavage site and extend our understanding of the cleavage site recognition process in general and the unusual cleavage of the tRNA(His) precursor in particular. Based on our as well as the data of others, we suggest that the nucleotide immediately upstream of the cleavage site is a positive determinant for cleavage by RNase P in general and the expression of tRNA genes is influenced by structural elements localized outside the promoter region i.e. in the leader and spacer regions of tRNA transcripts.

Metal ion cooperativity in ribozyme cleavage of RNA

Combinations of chemical and genetic approaches were used to study the function of divalent metal ions in cleavage of RNA by the ribozyme RNase P RNA. We show that different divalent metal ions have differential effects on cleavage site recognition and rescue of cleavage activity by mixing divalent metal ions that do not promote cleavage by themselves. We conclude that efficient and correct cleavage is the result of cooperativity between divalent metal ions bound at different sites in the RNase P RNA-substrate complex. Complementation of a mutant RNase P RNA phenotype as a result of divalent metal ion replacement is demonstrated also. This finding together with other data indicate that one of the metal ions involved in this cooperativity is positioned near the cleavage site. The possibility that the Mg2+/Ca2+ ratio might regulate the activity of biocatalysts that depend on RNA for activity is discussed.

Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site

Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5′ termini by cleaving tRNA precursors immediately 5′ of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNAGln G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNAGln and pre-tRNATyrSu3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+72 influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNAGln and pre-tRNATyrSu3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.