Recognition of a Pre-tRNA Substrate by the Bacillus subtilis RNase P Holoenzyme

• Published in: Biochemistry (Easton) Vol. 37; no. 44; pp. 15466 - 15473
• Main Authors: LORIA, Andrew, NIRANJANAKUMARI, S., FIERKE, Carol A., PAN, Tao
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.11.1998
• Subjects: Bacillus subtilis; Bacillus subtilis - enzymology; Base Sequence; Binding Sites - genetics; DNA Footprinting; Edetic Acid - metabolism; Endoribonucleases - genetics; Endoribonucleases - metabolism; Ferrous Compounds - metabolism; Holoenzymes - genetics; Holoenzymes - metabolism; Hydrogen-Ion Concentration; Hydrolysis; Molecular Sequence Data; Protein Binding - genetics; Ribonuclease P; RNA Precursors - genetics; RNA Precursors - metabolism; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; RNA, Transfer, Phe - genetics; RNA, Transfer, Phe - metabolism; Substrate Specificity - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9816507

The holoenzyme of the bacterial RNase P has broader selectivity for biological substrates compared to the RNA alone (denoted P RNA) reaction. The structural basis of the substrate selectivity is investigated using a pre-tRNA substrate containing single-atom modifications by single turnover kinetics. Hydroxyl radical protection of the holoenzyme in the absence of the substrate shows that the RNase P protein binds to several regions in P RNA. The holoenzyme interacts with a subset of functional groups in the T stem-loop region of a pre-tRNA substrate previously identified to directly contact P RNA. The subtle change in structural recognition allows the holoenzyme to recognize RNA structures with only a small perturbation in an A-form helix at the corresponding position of the T stem-loop. This altered profile may permit the holoenzyme to bind non-tRNA substrates with little change in catalytic efficiency. The holoenzyme recognizes the same set of functional groups as the P RNA reaction in the region around the cleavage site and shows similar cleavage site selection compared to the P RNA reaction. These results suggest that the holoenzyme does not alter the fundamental mechanism of this enzymatic reaction. Rather, the holoenzyme significantly affects the binding affinity of an RNA substrate through additional interactions with the 5' leader [Kurz, C. A., Niranjanakumari, S., and Fierke, C. A. (1998) Biochemistry 37, 2393] and through altered recognition of the substrate structure.