Biomedical subjects

Victoria G Kolupaeva

Publications and source records attributed to Victoria G Kolupaeva.

6 recordsLinked to original sources

PubMed2006

Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex.

Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CCAUGG, in which the purines at (-)3 and (+)4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon-anticodon base-pairing during 48S initiation complex formation, and that the role of (-)3 and (+)4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at (+)4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C(1696) and less well to AA(1818-1819) in helix 44 of 18S rRNA, G cross-linked exclusively to AA(1818-1819). U at (-)3 cross-linked to rpS5 and eIF2alpha, whereas G cross-linked only to eIF2alpha. Results of UV cross-linking experiments and of assays of 48S complex formation done using alpha-subunit-deficient eIF2 indicate that eIF2alpha's interaction with the (-)3 purine is responsible for recognition of the (-)3 context position by 43S complexes and suggest that the (+)4 purine/AA(1818-1819) interaction might be responsible for recognizing the (+)4 position.

Animals↗

PubMed2005

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association.

The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j-, respectively). Although unlike eIF3j+, eIF3j- does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2*GTP*Met-tRNA(i)Met ternary complex, eIF3j-/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j- nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.

Eukaryotic Initiation Factor-3↗

PubMed2003

Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing.

Eukaryotic initiation factor (eIF) eIF1 maintains the fidelity of initiation codon selection by enabling 43S complexes to reject codon-anticodon mismatches, to recognize the initiation codon context, and to discriminate against establishing a codon-anticodon interaction with AUGs located <8 nt from the 5'-end of mRNA. To understand how eIF1 plays its discriminatory role, we determined its position on the 40S ribosomal subunit using directed hydroxyl radical cleavage. The cleavage of 18S rRNA in helices 23b, 24a, and 44 by hydroxyl radicals generated from Fe(II) tethered to seven positions on the surface of eIF1 places eIF1 on the interface surface of the platform of the 40S subunit in the proximity of the ribosomal P-site. The position of eIF1 on the 40S subunit suggests that although eIF1 is unable to inspect the region of initiation codon directly, its position close to the P-site is very favorable for an indirect mechanism of eIF1's action by influencing the conformation of the platform of the 40S subunit and the positions of mRNA and initiator tRNA in initiation complexes. Unexpectedly, the position of eIF1 on the 40S subunit was strikingly similar to the position on the 30S ribosomal subunit of the sequence and structurally unrelated C-terminal domain of prokaryotic initiation factor IF3, which also participates in initiation codon selection in prokaryotes.

Animals↗

PubMed2003

Mapping the binding interface between human eukaryotic initiation factors 1A and 5B: a new interaction between old partners.

The translation initiation factors (IFs) IF1/eIF1A and IF2e/IF5B have been conserved throughout all kingdoms. Although the central roles of the bacterial factors IF1 and IF2 were established long ago, the importance of their eukaryotic homologs, eukaryotic IFs (eIFs) eIF1A and eIF5B, has only recently become evident. The translation machinery in eukaryotes is more complex and accordingly, eIF1A and eIF5B seem to have acquired a number of new functions while also retaining many of the roles of bacterial IF1 and IF2. IF1 and IF2 have been shown to interact on the ribosome but no binding has been detected for the free factors. In contrast, yeast eIF1A and eIF5B have been reported to interact in the absence of ribosomes. Here, we have identified the binding interface between human eIF1A and the C-terminal domain of eIF5B by using solution NMR. That interaction interface involves the C termini of the two proteins, which are not present in bacterial IF1 and IF2. The interaction is, therefore, unique to eukaryotes. A structural model for the interaction of eIF1A and eIF5B in the context of the ribosome is presented. We propose that eIF1A and eIF5B simultaneously interact at two sites that are >50 A apart: through their C termini as reported here, and through an interface previously identified in bacterial IF1 and IF2. The binding between the C termini of eIF1A and eIF5B has implications for eukaryote-specific mechanisms of recruitment and release of translation IFs from the ribosome.

Binding Sites↗

PubMed2003

Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site.

Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG834). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3' border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG834 for productive binding of the ribosome.

Adenosine Triphosphate↗

PubMed2002

The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection.

To elucidate an outline of the mechanism of eukaryotic translation initiation, 48S complex formation was analyzed on defined mRNAs in reactions reconstituted in vitro from fully purified translation components. We found that a ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3, and the eIF2 ternary complex form a 43S complex that can bind to the 5'-end of an unstructured 5'-untranslated region (5'-UTR) and in the presence of eIF1 scan along it and locate the initiation codon without a requirement for adenosine triphosphate (ATP) or factors (eIF4A, eIF4B, eIF4F) associated with ATP hydrolysis. Scanning on unstructured 5'-UTRs was enhanced by ATP, eIFs 4A and 4B, and the central domain of the eIF4G subunit of eIF4F. Their omission increased the dependence of scanning on eIFs 1 and 1A. Ribosomal movement on 5'-UTRs containing even weak secondary structures required ATP and RNA helicases. eIF4F was essential for scanning, and eIFs 4A and 4B were insufficient to promote this process in the absence of eIF4F. We report that in addition to its function in scanning, eIF1 also plays a principal role in initiation codon selection. In the absence of eIF1, 43S complexes could no longer discriminate between cognate and noncognate initiation codons or sense the nucleotide context of initiation codons and were able to assemble 48S complexes on 5'-proximal AUG triplets located only 1, 2, and 4 nt from the 5'-end of mRNA.

5' Flanking Region↗