ABSTRACT
Possible involvement of 18S rRNA fragment 1638-1650 including basements of the helices h44 and h28 and nucleotides of the ribosomal decoding site in the cap-independent translation initiation on plant ribosomes is studied. This rRNA fragment is shown to be accessible for complementary interactions within the 40S ribosomal subunit. It is found that the sequence complementary to the 18S rRNA fragment 1638-1650 is able to enhance efficiency of a reporter mRNA translation when placed just after the initiation codon. The results obtained indicate that in the course of the cap-independent translation initiation, complementary interactions can occur between mRNA coding sequence and 18S rRNA fragment in the region of the ribosomal decoding site.
Subject(s)
Gene Expression Regulation, Plant , Peptide Chain Initiation, Translational , RNA, Messenger/genetics , RNA, Ribosomal, 18S/genetics , Triticum/genetics , Base Pairing , Base Sequence , Codon, Initiator/genetics , Molecular Sequence Data , Nucleic Acid Conformation , Open Reading Frames , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/metabolism , Ribosome Subunits, Small, Eukaryotic , Triticum/metabolismABSTRACT
A possibility of involvement of 3'-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3-terminal segment (nucleotides 1777-1811) of 18S rRNA including the last hairpin 45 is accessible for complementary interactions in 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA when added to wheat germ cell-free protein synthesizing system were found to specifically inhibit translation of uncapped reporter mRNA coding for beta-glucuronidase, which bears in the 5'-untranslated region (UTR) a leader sequence of potato virus Y (PVY) genomic RNA possessing fragments complementary to the region 1777-1811. It was shown that a sequence corresponding to nucleotides 291-316 of PVY, which is complementary to a major portion of the 3-terminal 18S rRNA segment 1777-1808, when placed into 5'-UTR, is able to enhance translational efficiency of the reporter mRNAs. The results obtained suggest that complementary interactions between mRNA 5'-UTR and 18S rRNA 3'-terminal segment can take place in the course of cap-independent translation initiation.
Subject(s)
Peptide Chain Initiation, Translational/genetics , RNA, Ribosomal, 18S/metabolism , Triticum/metabolism , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Base Sequence , Glucuronidase/chemistry , Glucuronidase/genetics , Molecular Sequence Data , Oligonucleotides/chemistry , Oligonucleotides/genetics , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Ribosomal, 18S/genetics , Ribosomes/chemistry , Ribosomes/genetics , Seeds/genetics , Seeds/metabolism , Triticum/geneticsABSTRACT
The binding of the 18S RNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112-1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5'-untranslated region upstream of the reporter sequence coding for beta-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5' end allowed for the demonstration that the 1112-1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112-1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences.
Subject(s)
RNA, Plant/physiology , RNA, Ribosomal, 18S/physiology , Ribosome Subunits, Small, Eukaryotic/metabolism , Triticum/metabolism , Enhancer Elements, Genetic , Genes, Reporter , Glucuronidase/biosynthesis , Glucuronidase/genetics , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/biosynthesis , Nucleic Acid Heteroduplexes/genetics , Oligodeoxyribonucleotides/chemistry , Potyvirus/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/chemistry , RNA, Ribosomal, 18S/chemistry , Seeds/metabolismABSTRACT
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 RaysABSTRACT
Ribosomal protein SA (rpSA) or p40 is a structural element of the small subunit of the eukaryotic ribosome. N-terminal and central parts of the protein are homologous to prokaryotic rpS2 whereas its C-terminal part is eukaryote specific. In this study we showed that samples of 40S ribosomal subunits isolated from full-term human placenta are variably deficient in the rpSA content. To reveal rpSA ability to bind to human 40S ribosomal subunits, recombinant rpSA and its mutant forms with N- and C-terminal deletions have been synthesized. It was shown that both full-size and truncated from the N-terminus proteins were able to bind to the 40S subunits whereas the mutant truncated from C-terminus was not.
