The Protein Component of Bacillus subtilis Ribonuclease P Increases Catalytic Efficiency by Enhancing Interactions with the 5‘ Leader Sequence of Pre-tRNAAsp

• Published in: Biochemistry (Easton) Vol. 37; no. 26; pp. 9409 - 9416
• Main Authors: CRARY, Sharon M., NIRANJANAKUMARI, S., FIERKE, Carol A.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 30.06.1998
• Subjects: Bacillus subtilis - enzymology; Binding Sites; Catalysis; Coenzymes - chemistry; Coenzymes - metabolism; Endoribonucleases - chemistry; Endoribonucleases - metabolism; Hydrolysis; Kinetics; Ribonuclease P; Ribonucleoproteins - chemistry; Ribonucleoproteins - metabolism; RNA Precursors - chemistry; RNA Precursors - metabolism; RNA, Bacterial - chemistry; RNA, Bacterial - metabolism; RNA, Catalytic - chemistry; RNA, Catalytic - metabolism; RNA, Transfer, Asp - chemistry; RNA, Transfer, Asp - metabolism; Substrate Specificity; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi980613c

Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the formation of the mature 5' end of tRNA. To investigate the role of the protein component in enhancing the affinity of Bacillus subtilis RNase P for substrate (Kurz, J. C., Niranjanakumari, S., Fierke, C. A. (1998) Biochemistry 37, 2393), the kinetics and thermodynamics of binding and cleavage were analyzed for pre-tRNAAsp substrates containing 5' leader sequences of varying lengths (1-33 nucleotides). These data demonstrate that the cleavage rate constant catalyzed by the holoenzyme is not dependent on the leader length; however, the association rate constant for substrate binding to holoenzyme increases as the length of the leader increases, and this is reflected in enhanced substrate affinity of up to 4 kcal/mol. In particular, the protein component of RNase P stabilizes interactions with nucleotides at -2 and -5 in the 5' leader sequence of the pre-tRNA substrate. A 1 nucleotide leader decreases substrate affinity >/=15-fold compared to tRNAAsp due to ground-state destabilization of the enzyme-substrate complex. This destabilization is overcome by increasing the length of the leader to 2 nucleotides due to P RNA-pre-tRNA contacts that are stabilized by the P protein. The affinity of RNase P holoenzyme (but not RNA alone) for pre-tRNAAsp is further enhanced with a substrate containing a 5 nucleotide leader. These data indicate that novel direct or indirect interactions occur between the 5' leader sequence of pre-tRNAAsp and the protein component of RNase P.