The path of mRNA through the bacterial ribosome: a site-directed crosslinking study using new photoreactive derivatives of guanosine and uridine.
Two new photoreactive nucleotide derivatives have been applied in site-directed crosslinking studies with mRNA analogues. 6-Thioguanosine triphosphate or 5-methyleneaminouridine triphosphate was incorporated into mRNA analogues by T7 transcription; after transcription, the 5-methyleneaminouridine residues were converted to a diazirine derivative. mRNA analogues carrying either 6-thioguanosine or the diazirine derivative were bound to Escherichia coli ribosomes in the presence of tRNA(f)(Met), and photo-crosslinking was induced by irradiation at 350 nm. With 6-thioguanosine, specific crosslinks were observed from downstream positions +8 or +9 of the mRNA to nt 1196 in helix 34 of the 16S rRNA, and from position +12 to nt 530 in helix 18. With the diazirine derivative, a crosslink from position +2 (within the AUG codon) to nt 926 in helix 28 was found. Taken together with previous data obtained from downstream sites in mRNA analogues carrying 4-thiouridine residues, specific crosslinks have now been identified from downstream mRNA positions +2, +4, +6, +7, +8, +9, +11, and +12. The data confirm that the three 16S rRNA regions involved-helices 18, 28, and 34-are in the direct neighborhood of the decoding area of the 30S subunit.
Sergiev
P V
PV
Department of Chemistry, Moscow State University, Russia.
Lavrik
I N
IN
Wlasoff
V A
VA
Dokudovskaya
S S
SS
Dontsova
O A
OA
Bogdanov
A A
AA
Brimacombe
R
R
D017123:Bacteriophage T7 / Q000378:metabolism; D001483:Base Sequence; D003062:Codon; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000096:biosynthesis* / Q000737:chemistry; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013873:Thionucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism
C105886:5-methyleneaminouridine triphosphate; D003062:Codon; D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012358:RNA, Transfer, Met; D013873:Thionucleotides; C032827:6-thioguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D014544:Uridine Triphosphate