ABSTRACT
The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotide G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementarily addressed modification of the corresponding RNA transcript with alkylating oligodeoxynucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that, in the binary complex of HCV IRES elements with the small subunit of the 80S ribosome, its subdomain IIId stem is located on the outer subunit surface between the head and the body next to the "beak" near the exit of mRNA from the ribosome.
Subject(s)
Hepacivirus/genetics , Models, Molecular , RNA, Viral/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Base Sequence , Cross-Linking Reagents/chemistry , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , RNA, Ribosomal, 18S/metabolism , RNA, Viral/chemistry , RNA, Viral/radiation effects , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/radiation effects , Ultraviolet RaysSubject(s)
Placenta/chemistry , RNA, Messenger/chemistry , Ribosomal Proteins/chemistry , Alkylation , Female , Humans , Pregnancy , Ribosomes/chemistryABSTRACT
The in vitro binding of total ribosomal proteins with rough endoplasmic membranes, from which 70% of ribosomes are eliminated by EDTA (ME) is studied. It is found that in conditions of specific interaction of ribosomes with membranes about 75% of total ribosomal proteins are bound with ME. Membranes, heterogenous in their content (different protein/lipid ratio), became homogenous in their buyoant density after the binding with proteins. The ability of membrane-ribosomal protein complex to bind ribosomes is not decreased, as it can be expected, but is considerablly increased, thus indicating on a non-specific character of ribosome binding. Ribosomal subunits lacking about half of structural protein are capable to bind with ribosome-binding membrane receptors and with some additional sites. This binding is also non-specific, because the binding efficiency of large and small subunits is the same.
