Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.
RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III.
Department of Life Sciences (BK21 program), Chung-Ang University, Seoul, Korea.
Kyungsub
K
Kim
Se-Hoon
SH
Sim
Che Ok
CO
Jeon
Younghoon
Y
Lee
Kangseok
K
Lee
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D043244:Ribonuclease III; C468366:ribonuclease III, E coli