PMC

A248 mutant cleavage specificity as a function of substrate base composition. (A) Fraction correct cleavage by native and A248 mutant ribozymes for substrates that were varied at N(-1). The fraction correct cleavage of dA(-1) is shown with white bars; dG(-1) with black bars; dC(-1) with gray bars; and dU(-1) with cross-hatched bars. Error bars indicate the standard deviation of three or more independent measurements. (B) Miscleavage as a function of N(-2) base identity, for substrates with dA at N(-1). White bars indicate the fraction of miscleavage for A(-2), black bars G(-2), gray bars C(-2), and cross-hatched bars U(-2).

Rescue of A248 mutant binding defects by N(-1) mutation. (A) The plot shows binding data for the native RNase P RNA with U(-1) substrate (diamonds); A248U binding U(-1) substrate (triangles); native RNase P RNA binding A(-1) substrate (squares); and A248U with A(-1) substrate (circles). (B) The plot shows binding data for native RNase P RNA with U(-1) substrate (diamonds); A248C binding U(-1) substrate (triangles); native RNase P RNA binding G(-1) substrate (squares); and A248C binding G(-1) substrate (circles). All experiments contain 2 nM final substrate concentrations and are fit to Equation 1.

Rescue of A248U miscleavage by N(-1) mutation. Annealed substrates (see Materials and Methods) with U, A, dU, or dA at N(-1) were cleaved overnight with the indicated A248 mutant ribozymes (lanes 8–23). Lanes 1 and 24 are size standards produced by partial ribonuclease T1 digestion of substrates. Lanes 2 and 25 are products from partial alkaline hydrolysis. Lane 3 contains oligonucleotides corresponding to products of correct (AAAAU) and incorrect (AAAA) cleavage. Lanes 4–7 show uncut substrate oligonucleotides. Variations in mobility of the correctly cut fraction are due to differences in nucleotide composition.

Cleavage of pre-tRNA bearing a 2′ deoxy modification at N(-1) by the native ribozyme and J5/15 mutants. Mobility of the correct cleavage product (AAAAU) is indicated. A248 mutants (lanes 6–8) show miscleavage to yield shorter 5′ products. A249G and A249C mutant enzymes do not cut to completion, but when quantified are observed to choose the correct cleavage site at the same frequency as the native enzyme. The presence of the band marked by an asterisk varied between substrate preparations and was not observed with annealed substrates (see Materials and Methods).