ABSTRACT
The decoding center of the ribosome provides mRNA translation and the fidelity of the codon--anticodon interactions along with mRNA translocation in the course of protein biosynthesis. The three-dimensional structure of the ribosome decoding center is still unknown. However, up to now a number of direct and indirect experimental data on the structural and functional organization of the decoding center have been obtained. In this paper the main components of the decoding center are described on the basis of our own experimental results combined with data from the literature. A model of their spatial arrangement at the small ribosomal subunit is suggested.
Subject(s)
RNA, Ribosomal/metabolism , Ribosomes/metabolism , Ribosomes/ultrastructure , Base Sequence , Models, Molecular , Models, Structural , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/metabolism , Protein Biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/genetics , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolismABSTRACT
Telomerase is a ribonucleoprotein responsible for maintaining telomeres during the cell cycle [1,2]. Here we describe a two-step purification procedure for the Saccharomyces cerevisiae telomerase complex. We have found that the properties (processivity, nuclease activity) of telomerase depend on the isolation procedure. Using a cross-linking approach, we have revealed several proteins that could be components of the telomerase complex. Furthermore, spectra of cross-linked proteins differ in processive and non-processive telomerase complexes.
Subject(s)
Fungal Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Telomerase/metabolism , Antisense Elements (Genetics) , Chromatography, Liquid/methods , Cross-Linking Reagents , DNA Primers/chemistry , DNA Primers/metabolism , Models, Chemical , Repetitive Sequences, Nucleic Acid , Telomerase/chemistry , Telomerase/isolation & purification , UltracentrifugationABSTRACT
A new approach for inserting a photo-label at a selected position within the long ribosomal RNA molecules has been developed. The Escherichia coli 16S rRNA was cleaved at a single internucleotide bond, 1141-1142, with RNase H in the presence of a complementary chimeric oligonucleotide. 4-Thiouridine 5', 3'-diphosphate was ligated to the 3'-end of the 5'fragment at the cleavage site with T4 RNA ligase. The 16S rRNA fragments containing this added photo-reactive nucleotide were assembled together with total 30S ribosomal proteins into small ribosomal subunits. The ability of such 30S particles containing fragmented rRNA to form 70S ribosomes has been demonstrated previously. Crosslinks were induced within the 30S subunits by mild UV irradiation. The sites of crosslinking within the 16S rRNA were then analyzed using RNase H digestion and reverse transcription. Two crosslinks from the thio-nucleotide attached to nt C1141 of 16S rRNA were observed, namely to nt U1295 and G1272. These results are in agreement with the established proximity of helix 39 and 41 in the 3D structure of the 30S ribosomal subunit, as shown by other intra RNA crosslinking data. These data furthermore allow us to refine the structural arrangement of helices 41 and 39 relative to one another.
Subject(s)
Escherichia coli/metabolism , Nucleic Acid Conformation , Oligoribonucleotides/chemical synthesis , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/metabolism , Ribosomes/metabolism , Bacteriophage T4/enzymology , Base Sequence , Chimera , Cross-Linking Reagents , Indicators and Reagents , Models, Molecular , Molecular Sequence Data , Oligoribonucleotides/chemistry , RNA Ligase (ATP) , RNA-Directed DNA Polymerase , Ribonuclease H , Substrate Specificity , Ultraviolet Rays , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate/chemical synthesisABSTRACT
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.
Subject(s)
Escherichia coli/metabolism , Guanosine Triphosphate/analogs & derivatives , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/biosynthesis , Ribosomes/metabolism , Thionucleotides/metabolism , Uridine Triphosphate/analogs & derivatives , Bacteriophage T7/metabolism , Base Sequence , Codon , Cross-Linking Reagents , Guanosine Triphosphate/chemical synthesis , Guanosine Triphosphate/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Bacterial/biosynthesis , RNA, Bacterial/chemistry , RNA, Messenger/biosynthesis , RNA, Messenger/chemistry , RNA, Ribosomal, 16S/chemistry , RNA, Transfer, Met/metabolism , Thionucleotides/chemical synthesis , Transcription, Genetic , Uridine Triphosphate/chemical synthesis , Uridine Triphosphate/metabolismABSTRACT
Telomeres, the natural ends of linear eukaryotic chromosomes, are essential for protecting chromosomes from degradation and fusion. The synthesis of telomere DNA repeats in most eukaryotes is performed by a special enzyme, telomerase. Telomerase, a ribonucleoprotein enzyme, is a specialized reverse transcriptase utilizing its RNA moiety as a template for synthesis of telomeric DNA. Enzymatic properties and results of comparative analysis of telomerase RNA and protein structures from different eukaryotic systems are discussed in this review.
Subject(s)
RNA/chemistry , RNA/metabolism , Telomerase/chemistry , Telomerase/metabolism , Animals , Base Sequence , DNA Replication , Nucleic Acid Conformation , Repetitive Sequences, Nucleic Acid , Substrate Specificity , Telomere/physiologyABSTRACT
5S rRNA is a small RNA molecule that is a component of a ribosome from almost all living organisms. In this review, we discuss the biogenesis of 5S rRNA and its properties as an independent structural domain of a ribosome as well as the current concepts concerning the higher order structure of 5S rRNA in free state and in its complexes with ribosomal proteins and its folding in the ribosome. Special attention is paid to recent experimental approaches that have been useful in 5S rRNA studies. Our own data on topography of 5S rRNA in the ribosomes are discussed in detail. The hypothesis describing the possible functional role of 5S rRNA for ribosome functioning is discussed.
Subject(s)
RNA, Ribosomal, 5S/genetics , Ribosomes/genetics , Base Sequence , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Ribosomal, 5S/chemistry , Ribosomal Proteins/chemistry , Structure-Activity RelationshipABSTRACT
Synthetic mRNA analogues were constructed with sequences related to the Cro-protein mRNA from lambda-phage and prepared by T7 transcription. Each mRNA contained several thiouridine (thio-U) residues. The regions upstream from the AUG initiator codon of the mRNA were the same in all the messages, whereas in the downstream part the thio-U residues were placed in selected positions. These positions covered the region from +4 to +16 (A in the initiator AUG codon being defined as +1). After binding to the ribosome in the presence of initiator tRNA the thio-U residues were activated by u.v. irradiation and the resulting sites of cross-linking to 16 S rRNA and ribosomal proteins were analysed. Cross-links to several ribosomal proteins were identified in different types of complex. Changes in the conformation of the small ribosomal subunit in different initiation and elongation complexes are discussed.
