The flexible arm of tRNase Z is not essential for pre-tRNA binding but affects cleavage site selection.
tRNase Z is an enzyme responsible for removing a 3' trailer from pre-tRNA. Although most tRNase Zs cleave pre-tRNAs immediately after the discriminator nucleotide with the exception of Thermotoga maritima tRNase Z, which cleaves after the (74)CCA(76) sequence, our knowledge was limited about how the cleavage site in pre-tRNA is selected. Bacterial tRNase Zs contain a unique domain termed flexible arm, which extends from the core domain. Using various tRNase Z variants, here we examined how the flexible arm affects the cleavage site selection. T. maritima tRNase Z variants with modified flexible arms shifted the cleavage site and a Bacillus subtilis tRNase Z variant with no flexible arm showed an anomalous cleavage activity. Some of the T. maritima/B. subtilis chimeric enzymes had both properties: they recognized (74)CCA(76)-containing pre-tRNA and cleaved it after the discriminator. Taken together, the present data indicate that the flexible arm is not essential for pre-tRNA binding but affects the cleavage site selection probably by pushing the distal region of the T arm in such a way that the discriminator nucleotide becomes closer to the catalytic site.
Minagawa
Asako
A
Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan.
Ishii
Ryohei
R
Takaku
Hiroaki
H
Yokoyama
Shigeyuki
S
Nashimoto
Masayuki
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D012322:RNA Precursors; D012343:RNA, Transfer; D004722:Endoribonucleases