Transfer RNA recognition by the Escherichia coli delta2-isopentenyl-pyrophosphate:tRNA delta2-isopentenyl transferase: dependence on the anticodon arm structure.
To elucidate the sequence elements required in the anticodon stem for the recognition of Escherichia coli tRNA(Ser) (GGA) by the E. coli isopentenyl-tRNA:A37 transferase (IPTT), which result in the conversion of A37 into isopentenylated i6A37, we have tested and characterized in vitro T7-runoff transcripts of 17 variants of E. coli tRNA(Ser)(GGA) and 7 other tRNAs from E. coli and yeast. Our results indicate that, instead of a stringent specific anticodon stem and loop sequence, the key feature required for the recognition of E. coli tRNAs by IPTT is the A36A37A38 sequence occurring within the seven-membered anticodon loop, and the retention of the standard helical structure and flexibility, especially in the proximal anticodon stem. The G30*U40 mismatch base pair close to the anticodon loop is strictly avoided. The frequent occurrence of a C-G base pair in the three stem locations closest to the loop (positions 29-41, 30-40 and 31-39) or the occurrence of even one such C-G base pair along with some other similarly less suited, but individually tolerated deviations can also totally abolish the A37 isopentenylation of tRNA. For the position 30-40, the G-C base pair is shown uniquely suited, whereas for the adjoining 29-41 stem location, a purine-pyrimidine base pair with pyrimidine on the 3'-side is strongly preferred. Retention of the overall 3D tRNA structure is favorable for isopentenylation and allows some tolerance of proximal stem sequence deviations. Our data suggest a recognition mode that implies the interaction of IPTT with the strictly conserved A36A37A38 sequence and the other functional groups located in the minor groove of the anticodon stem.
Motorin
Y
Y
C.N.R.S., Laboratoire d'Enzymologie et Biochimie Structurales, Gif-sur-Yvette, France.
Bec
G
G
Tewari
R
R
Grosjean
H
H
D019883:Alkyl and Aryl Transferases; D000926:Anticodon / Q000737:chemistry* / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D017124:Conserved Sequence; D016208:Databases, Factual; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007541:Isopentenyladenosine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012362:RNA, Transfer, Ser / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014166:Transferases / Q000378:metabolism*
D000926:Anticodon; D012362:RNA, Transfer, Ser; D007541:Isopentenyladenosine; D012343:RNA, Transfer; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase