Previous analyses of complexes 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by IRES proteins. Here, using chemical probing, we show that HCV also backbone and bases CCC triplet in 18S RNA (rRNA) expansion segment 7. These presumably provide interplay between domain II AUG codon close to protein S5, which causes a rearrangement rRNA structure vicinity universally conserved nucleotide G1639. As result, G1639 becomes...

The expression of ribosomal protein (rp) genes is regulated at multiple levels. In yeast, two are autoregulated by feedback effects the on pre-mRNA splicing. Here, we have investigated whether similar mechanisms occur in eukaryotes with more complicated and highly splicing patterns. Comparisons sequences S13 gene (RPS13) among mammals birds revealed that intron 1 conserved than other introns. Transfection HEK 293 cells a minigene-expressing showed presence reduced factor four. Ribosomal was...

The aim of the present study was to investigate homoharringtonine alkaloid effect on: (i) nonenzymatic and eEF-1-dependent Phe-tRNAPhe binding poly(U)-programmed human placenta 80 S ribosomes; (ii) diphenylalanine synthesis accompanying binding; (iii) acetylphenylalanyl-puromycin formation. Neither nor were noticeably affected by alkaloid, whereas puromycin reaction strongly inhibited homoharringtonine. It has been proposed that site on ribosomes should overlap or coincide with acceptor ribosome.

Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient following structural studies RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry...

Reverse‐phase HPLC was used to fractionate 40S ribosomal proteins from human placenta. Application of a C 4 , reverse‐phase column allowed us obtain 27 well‐resolved peaks. The protein composition each chromatographic fraction established by two‐dimensional polyacrylamide gel electrophoresis and N‐terminal sequencing. N‐terminally blocked were cleaved with endoproteinase Lys‐C, suitable peptides sequenced. All sequences compared those available data bases. This identify all the subunit in...

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding requires complex mechanism, comprising cis-acting SECIS RNA hairpin in 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, Binding Protein 2 (SBP2) central to mechanism. SBP2 has been so far functionally characterized only rats humans. In this work, we report characterization Drosophila melanogaster (dSBP2). Despite its shorter length, it retained same synthesis-promoting...

To study positioning of the polypeptide release factor eRF1 toward a stop signal in ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives oligoribonucleotides. The human peptides cross-linked to these short mRNAs were identified. Cross-linkers on guanines at second, third, and fourth positions modified fragment 31–33, lesser extent amino acids within region 121–131 (the “YxCxxxF loop”) N domain. Hence, both regions are involved recognition purines. A cross-linker...

Site-directed spin labeling (SDSL) is widely applied for structural studies of biopolymers by electron paramagnetic resonance (EPR). However, SDSL long RNA sequences still remains a challenging task. Here, we propose novel approach potentially suitable natural RNAs, which based on the attachment linker containing an aliphatic amino group to target nucleotide residue followed selective coupling label this group. Such can be attached desired via sequence-specific reaction with derivatives...

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive analogs were applied. Derivatives UUCUAAA heptaribonucleotide containing UUC codon for Phe UAAA, which bore a perfluoroaryl azido group at either fourth nucleotide or 3′‐terminal phosphate, synthesized. The was directed P site by cognate tRNA , targeting UAA A site. Mild UV irradiation ternary complexes consisting ribosome,...

Messenger RNA analogues (42-mers) containing a GAC codon (aspartic acid) in the middle of their sequence followed by s4UGA stop were used to identify components human ribosomal A site direct contact with photoactivatable 4-thiouridine (s4U) residue. We compared behavior nonphased ribosome−mRNA complex, (−)tRNAAsp, one phased (+)tRNAAsp, absence and presence eRF1, eukaryotic class 1 translation termination factor origin. The patterns cross-links obtained for three complexes similar those...

Abstract Previously we have shown that the CCA end of a P‐tRNA can be crosslinked with RPL36AL protein large subunit mammalian ribosomes; it belongs to L44e family present in all eukaryotic and archaeal ribosomes. Here confirm extend this finding demonstrate that: 1) crosslink is specific for tRNA at P/E hybrid site, as other positions pre‐translocational ribosomes could not ribosomal protein, 2) was formed most efficiently C74 C75 P/E‐tRNA, but also connect ultimate A Lys53 RPL36AL, 3)...

The amino acid selenocysteine is encoded by UGA, usually a stop codon, thus requiring specialized machinery to enable its incorporation into selenoproteins. comprises the tRNA Sec , 3′-UTR mRNA stem–loop termed SElenoCysteine Insertion Sequence (SECIS), which mandatory for recoding UGA as SECIS Binding Protein 2 (SBP2), and other proteins. Little known about molecular mechanism and, in particular, when, where, how SBP2 contact ribosome. Previous work others used isolated RNA address this...

SBP2 is a pivotal protein component in selenoprotein synthesis. It binds the SECIS stem–loop 3′ UTR of mRNA and interacts with both specialized translation elongation factor ribosome at 60S subunit. In this work, our goal was to identify binding partners on ribosome. Cross-linking experiments bifunctional reagents demonstrated that SBP2-binding site human mainly formed by 28S rRNA. Direct hydroxyl radical probing entire rRNA revealed bound 80S ribosomes or subunits protects helix ES7L-E...

Affinity labeling of human placental 80S ribosomes with mRNA analogs up to 12 uridyl residues, i.e. alkylating derivatives oligouridylates bearing either 4‐( N ‐2‐chloroethyl‐ ‐methyl‐amino)benzylmethylphosphamide group at the 5′‐termini or 2′,3′‐O‐[4‐( ‐methyl‐amino)]benzylidene residue attached 3′‐termini, in presence cognate Phe‐tRNA Phe has been investigated. All modified only 40S subunit. The fraction 18S rRNA by 5′‐end decreased dramatically extension reagent oligouridylate moiety....

Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone UAAA stop signal in ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing UAA/UAAA a photoactivatable cross-linker at definite locations. The human peptides cross-linked to these were identified. Cross-linkers on 2nd, 3rd or 4th position modified near conserved YxCxxxF loop (positions 125–131 N domain), but mainly tripeptide 26-AAR-28. This also with derivatized 3′-phosphate UAA,...

Protein uL2 is essential for the catalytic activity of ribosome and has a conserved shape in ribosomes from all domains life. However, sequence its unstructured C-terminal loop apex that contacts 23S/28S rRNA helix (H) 93 near ribosomal peptidyl transferase center differs bacteria, archaea eukaryotes. Eukaryote-specific residue His216 located this mammalian hydroxylated ribosomes. We used set chemical probes to explore structure an RNA mimicked segment 28S domain V containing part binding...