Figure 4.

Titration of RNA. A, 4-fold surplus of YvcI (monomer) over the RNA substrate is needed for efficient catalysis. ppp-RNA (AGU 5S rRNA, 5′-labeled) was incubated with YvcI (2 μm) in 1× manganese buffer (2 mm Mn²⁺). The RNA–enzyme ratio is as indicated. PEI-cellulose TLC, phosphorimaging of radioactivity is shown. B and C, quantification of ppp-RNA, p_i, and pp_i from A. B, quantification of the cleavage products at an RNA–enzyme ratio of 1:4, showing means ± S.D. of two independent experiments (technical replicates). C, quantification of p_i and pp_i. The RNA–enzyme ratio is indicated. Statistics are as for B. D, multiangle light-scattering analysis indicates YvcI to be a tetramer in solution. The scattered light (gray) and the protein concentration (blue) were used to determine the molar mass distribution (red) along the YvcI elution peak.