Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously ...demonstrated that a hybrid eRF1 carrying the
Euplotes octocarinatus domain 1 fused to
Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Two exceptional mechanisms of eukaryotic translation initiation have recently been identified that differ fundamentally from the canonical factor-mediated, end-dependent mechanism of ribosomal ...attachment to mRNA. Instead, ribosomal 40S subunits bind in a factor-independent manner to the internal ribosomal entry site (IRES) in an mRNA. These two mechanisms are exemplified by initiation on the unrelated ∼300 nt.-long Hepatitis C virus (HCV) IRES and the ∼200 nt.-long cricket paralysis virus (CrPV) intergenic region (IGR) IRES, respectively. Ribosomal binding involves interaction with multiple non-contiguous sites on these IRESs, and therefore also differs from the factor-independent attachment of prokaryotic ribosomes to mRNA, which involves base-pairing to the linear Shine–Dalgarno sequence. The HCV IRES binds to the solvent side of the 40S subunit, docks a domain of the IRES into the ribosomal exit (E) site and places the initiation codon in the ribosomal peptidyl (P) site. Subsequent binding of eIF3 and the eIF2-GTP/initiator tRNA complex to form a 48S complex is followed by subunit joining to form an 80S ribosome. The CrPV IRES binds to ribosomes in a very different manner, by occupying the ribosomal E and P sites in the intersubunit cavity, thereby excluding initiator tRNA. Ribosomes enter the elongation stage of translation directly, without any involvement of initiator tRNA or initiation factors, following recruitment of aminoacyl-tRNA to the ribosomal aminoacyl (A) site and translocation of it to the P site.
To cite this article: A.V. Pisarev et al., C. R. Biologies 328 (2005).
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The canonical initiation process is the most complex aspect of translation in eukaryotes. It involves the coordinated interactions of at least 11 eukaryotic initiation factors, 40S and 60S ribosomal ...subunits, mRNA, and aminoacylated initiator tRNA (Met-tRNA(i)(Met)), as well as binding and hydrolysis of GTP and ATP. The factor requirements for many individual steps in this process, including scanning, initiation codon recognition, and ribosomal subunit joining, have until recently been obscure. We established the factor requirements for these steps by reconstituting the initiation process in vitro from individual purified components of the translation apparatus and developed approaches to explain the mechanism of individual steps and the roles of individual factors and to characterize the structure of initiation complexes. Here we describe protocols for the purification of native initiation factors and for expression and purification of active recombinant forms of all single subunit initiation factors, for the reconstitution of the initiation process, and for determination of the position of ribosomal complexes on mRNA by primer extension inhibition ("toe printing"). We also describe protocols for site-directed ultraviolet (UV) cross-linking to determine the interactions of individual nucleotides in mRNA with components of the initiation complex and for directed hydroxyl radical probing to determine the position of initiation factors on the ribosome.
The results of the numerical simulations of the dynamics of shallow waters for Volga-Akhtuba Floodplain are discussed. The mathematical model is based on the system of Saint-Venant equations. ...Numerical solution applies a combined Lagrangian-Eulerian (cSPH-TVD) algorithm. We have investigated the features of the spring flood in 2011 and found the inapplicability of the hydrodynamical model with the constant roughness coefficient
n
M
. We have found a good agreement between the results of numerical simulations and the observational data at gauging stations which allows us to estimate
n
M
in low water
n
M
min
=
0.02
and the maximum water level in the river Volga
n
M
max
=
0.06–0.07.
Objectives - to analyze the cases of isolated lesions of the sphenoid sinuses and to identify the main errors in the differential diagnosis in the prehospital and treatment periods. Material and ...methods. The study includes the data on the treatment of 58 patients with an isolated lesion of the sphenoid sinus in the period 2015-2018. The patients' age varied from 18 to 68 years. Gender distribution: men - 21 (30.7%), women - 37 (69.3%). Results. The following surgical approaches were used: transnasal access, extended transnasal access, transetmoid access, access according to the Bolger Box technique. In one case, the transpterygoid approach was used. When performing extended access, it was possible to achieve the formation of persistent, epithelized anastomosis in all the cases. In polypous-purulent forms of sphenoiditis, the relapses of the disease were most often observed, however, only anti-inflammatory therapy was required, including a sinus irrigation through the formed anastomosis. Conclusion. Further studies are required, to examine the features/ safety and clinical efficacy of the various endosurgical approaches.
The surface of VT-22 Russian grade titanium alloy samples was modified by inductively coupled plasma (ICP) nitriding followed by magnetron deposition of TiN coatings. Different operating conditions ...of ICP nitriding and magnetron deposition were considered. The microhardness depth profiles were measured for samples after nitriding. The performance of TiN coatings was examined with a scratch tester.
TiN coatings were deposited on a new Al super-alloy by magnetron sputtering in argon nitrogen environment. The deposited layer structure, microhardness, adhesion, corrosion resistance, and fatigue ...life were investigated and tests demonstrated improved performance of the alloy.
Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation ...complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNA
Met
i attach to the 5′-proximal region of mRNA and then scan along the 5′ untranslated region (5′UTR) to the initiation codon. Attachment of 43S complexes is mediated by three other eIFs, 4F, 4A, and 4B, which cooperatively unwind the cap-proximal region of mRNA and later also assist 43S complexes during scanning. We now report that these seven eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5′UTRs, and that this process requires the DExH-box protein DHX29. DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes and is required for DHX29's activity in promoting 48S complex formation.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A method of analysis of translation initiation complexes by toeprinting has recently acquired a wide application to investigate molecular mechanisms of translation initiation in eukaryotes. So far, ...this very fruitful approach was used when researchers did not aim to discriminate between patterns of toeprints for 48S and 80S translation initiation complexes. Here, using cap-dependent and internal ribosomal entry site (IRES)-dependent mRNAs, we show that the toeprint patterns for 48S and 80S complexes are distinct whether the complexes are assembled in rabbit reticulocyte lysate or from fully purified individual components. This observation allowed us to demonstrate for the first time a delay in the conversion of the 48S complex into the 80S complex for β-globin and encephalomyocarditis virus (EMCV) RNAs, and to assess the potential of some 80S antibiotics to block polypeptide elongation. Besides, additional selection of the authentic initiation codon among three consecutive AUGs that follow the EMCV IRES was revealed at steps subsequent to the location of the initiation codon by the 40S ribosomal subunit.
Full text
Available for:
BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the ...molecular mechanisms underlying these phenomena using the in vitro assembly of 48 S preinitiation complexes from fully purified translational components in the presence or absence of p50 as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3, and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for comparison) strongly stimulates formation of the 48 S preinitiation complexes with β-globin mRNA. This stimulation is observed when just a few molecules of p50 are bound per molecule of the mRNA. When the amount of p50 in solution is increased over some threshold p50/mRNA ratio, a remarkable repression is observed that can still be relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50, the inhibitory effect becomes irreversible. The threshold ratio depends upon the extent of secondary structure of the 5′-untranslated region linked to the β-globin coding region. Chemical probing has confirmed that the binding of p50 to mRNA involves only the sugar-phosphate backbone of the mRNA leaving nucleotide bases free for interaction with other messenger ribonucleoprotein (mRNP) components. These data are best compatible with the functional role of p50 as a “manager” of mRNA-protein interactions in mammalian mRNPs.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PDF
