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The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carring several photoreactive labels simutaneously on either side of the AUG start codonстатья
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Дата последнего поиска статьи во внешних источниках: 20 ноября 2019 г.
- Авторы: Dontsova O., Kopylov A., Brimacombe R.
- Журнал: EMBO Journal
- Том: 10
- Год издания: 1991
- Издательство: Nature Publishing Group
- Местоположение издательства: United Kingdom
- Первая страница: 2613
- Последняя страница: 2620
- DOI: 10.1002/j.1460-2075.1991.tb07803.x
- Аннотация: Messenger RNA molecules 30-35 bases long, with sequences related to the 5'-region of cro-mRNA from lambda-phage, were prepared by T7 transcription from synthetic DNA templates. Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio-UTP. Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross-linking of the thio-U residues was induced by UV irradiation at wavelengths greater than 300 nm. The cross-linked ribosomal proteins were identified immunologically, and cross-linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides. In both cases, the particular thio-U residue involved in the cross-link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross-linked complex. The principal results were that, at thio-U positions upstream of the AUG codon, specific cross-linking occurred to protein S7 and to the 3'-terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes. Less specific cross-linking was observed to proteins S1, S18 and S21 at various positions within the mRNA. Six bases downstream from the AUG codon, a tRNA-dependent cross-link was found to position approximately 1050 of the 16S RNA, but--in contrast to similar experiments with 70S ribosomes--no cross-linking was found to the 1390-1400 region.
- Добавил в систему: Донцова Ольга Анатольевна