Expanding the Genetic Code: Unnatural Base Pairs in Biological Systems

Molecular Biology - The genetic code is considered to use five nucleic bases (adenine, guanine, cytosine, thymine and uracil), which form two pairs for encoding information in DNA and two pairs for...     

Comparative chromosome painting

The review of the data on comparative chromosomal painting in mammals is presented. The development of new molecular-cytogenetic methods has resulted in the accumulation of the detailed information on homology of chromosomal segments of more than 50 species from 11 orders. In this review, modern methods of obtaining painting probes are considered in detail, and the basic tendencies of karyotype evolution in different taxa are discussed. Putative karyotypes of the ancestors of primates, carnivores, and placental mammals are considered.     

HEMK-Like Methyltransferases in the Regulation of Cellular Processes

Molecular Biology - Human translational methyltransferase (methylase) HEMK2, whose orthologues are found in many prokaryotes and eukaryotes, methylates such diverse substrates as glutamine and...     

Stabilization of eukaryotic ribosomal termination complexes by deacylated tRNA

Stabilization of the ribosomal complexes plays an important role in translational control. Mechanisms of ribosome stabilization have been studied in detail for initiation and elongation of eukaryotic translation, but almost nothing is known about stabilization of eukaryotic termination ribosomal complexes. Here, we present one of the mechanisms of fine-tuning of the translation termination process in eukaryotes. We show that certain deacylated tRNAs, remaining in the E site of the ribosome at the end of the elongation cycle, increase the stability of the termination and posttermination complexes. Moreover, only the part of eRF1 recognizing the stop codon is stabilized in the A site of the ribosome, and the stabilization is not dependent on the hydrolysis of peptidyl-tRNA. The determinants, defining this property of the tRNA, reside in the acceptor stem. It was demonstrated by site-directed mutagenesis of tRNA^Val and construction of a mini-helix structure identical to the acceptor stem of tRNA. The mechanism of this stabilization is different from the fixation of the unrotated state of the ribosome by CCA end of tRNA or by cycloheximide in the E site. Our data allow to reveal the possible functions of the isodecoder tRNAs in eukaryotes.     

Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3

Translation termination in eukaryotes is governed by two proteins, belonging to the class-1 (eRF1) and class-2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to GDP and inorganic phosphate in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA and peptidyl-tRNA but needs the presence of eRF1. It's known that eRF1 and eRF3 interact with each other in vitro and in vivo via their C-terminal regions. eRF1 consists of three domains - N, M, and C. In this study we examined the influence of individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N-, M-, C- and NM-domains induces eRF3 GTPase activity in presence of the ribosomes. MC-domain does induce GTPase activity of eRF3 but four times less efficient than full-length eRF1, therefore, MC-domain (and very likely M-domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated, postulating that GTPase activity eRF3 during the translation termination is associated with the intermolecular interactions of GTP/GDP, GTPase center of the large ribosomal subunit (60S), MC-domain of eRF1, C-terminal region and GTP-binding domains of eRF3, but without participation of the N-terminal region of eRF3.     

Interface of the interaction of the middle domain of human translation termination factor eRF1 with eukaryotic ribosomes

Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2, polypeptide chain release factors with the ribosome. The middle (M) domain of the class 1 factor eRF1 contains the strictly conserved GGQ motif and is involved in hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the large ribosome subunit. Heteronuclear NMR spectroscopy was used to map the interaction interface of the M domain of human eRF1 with eukaryotic ribosomes. The protein was found to specifically interact with the 60S subunit, since no interaction was detected with the 40S subunit. The amino acid residues forming the interface mostly belong to long helix α1 of the M domain. Some residues adjacent to α1 and belonging to strand β5 and short helices α2 and α3 are also involved in the protein-ribosome contact. The functionally inactive G183A mutant interacted with the ribosome far more weakly as compared with the wild-type eRF1. The interaction interfaces of the two proteins were nonidentical. It was concluded that long helix α1 is functionally important and that the conformational flexibility of the GGQ loop is essential for the tight protein-ribosome contact.     

eIF3j facilitates loading of release factors into the ribosome

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions – binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.     

Translation termination factor eRF1 of the ciliate <Emphasis Type="Italic">Blepharisma japonicum</Emphasis> recognizes all three stop codons

In species with variant genetic codes, one or two stop codons encode amino acid residues and are not recognized by the intrinsic class I translation termination factor (eRF1). Ciliata include a large number of species with variant genetic codes. The stop codon specificity of the Blepharisma japonicum translation termination factor eRF1 was determined in an in vitro eukaryotic translation system and in an in vivo assay (a dual reporter system). It was shown that eRF1 of B. japonicum retained specificity to all three stop codons, although the efficiency of peptydyl-tRNA hydrolysis in the presence of UGA was reduced in the in vitro assay. Since Heterotrichea (including B. japonicum) are the earliest diverged lineage in the phylogenetic tree of ciliates, B. japonicum probably possesses a universal genetic code similar to the putative ciliate ancestor group.     

Translation termination factor of eRFI of the ciliate Blepharisma japonicum recognizes all three stop codons

We have determined the type of stop codon specificity of Blepharisma japonicum translation termination factor eRF1 in an in vitro reconstituted eukaryotic translation system and in in vivo assay (the dual reporter system). We have shown that B. japonicum eRF1 retained specificity towards all three stop codons although efficiency of peptydyl-tRNA hydrolysis in the presence of UGA is reduced in an in vitro assay. We suggest that since the heterotrich B. japonicum represents the earliest diverged lineage on phylogenetic tree of ciliates, B. japonicum has the universal genetic code as ancestor group for all ciliates.     

Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.