Correlation between processing efficiency for ribonuclease P minimal substrates and conformation of the nucleotide -1 at the cleavage position.
It is demonstrated that acceptor stem duplexes derived from native tRNAs which contain a three-nucleotide extension at the 5'-terminus of mature tRNA are minimal substrates for ribonuclease P from both Escherichia coli and Bacillus subtilis. Variants with a cytidine at position -1 are most efficiently processed whereas the G -1 variant represents a comparatively poor substrate. An A -1 acceptor stem variant is a slightly better substrate than the G -1 variant though generally distinctly less efficient than the C -1 duplex. This is in qualitative agreement with the frequency of the occurrence of the corresponding nucleotides at position -1 in natural substrates, which is highest for pyrimidines and least for G. NMR analyses of the corresponding acceptor stems reveal that the conformation of the nucleotides at position -1 correlates with the substrate preferences of Ribonuclease P: Whereas C -1 adopts a conformation characterized by a glycosidic angle in the anti range (close to high-anti), the G -1 is clearly in syn conformation, and that of A -1 is intermediate between high-anti and syn. The riboses of nucleotides -1 are in all cases predominantly 2'-endo puckered.
Zuleeg
T
T
Lehrstuhl für Biochemie, Universität Bayreuth, and Ribopharma AG, Universitätsstrasse 30, D-95440 Bayreuth, Germany.
Hansen
A
A
Pfeiffer
T
T
Schübel
H
H
Kreutzer
R
R
Hartmann
R K
RK
Limmer
S
S
D000241:Adenosine / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D001482:Base Composition; D003562:Cytidine / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006151:Guanosine / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D043262:Ribonuclease P; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D016337:RNA, Catalytic; D006151:Guanosine; D003562:Cytidine; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D000241:Adenosine