Biomedical subjects

Ludmila Frolova

Publications and source records attributed to Ludmila Frolova.

7 recordsLinked to original sources

PubMed2005

Invariant amino acids essential for decoding function of polypeptide release factor eRF1.

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125-131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.

Amino Acid Sequence↗

PubMed2005

The first position of a codon placed in the A site of the human 80S ribosome contacts nucleotide C1696 of the 18S rRNA as well as proteins S2, S3, S3a, S30, and S15.

Messenger RNA analogues (42-mers) containing a GAC codon (aspartic acid) in the middle of their sequence followed by a s(4)UGA stop codon were used to identify the components of the human ribosomal A site in direct contact with the photoactivatable 4-thiouridine (s(4)U) residue. We compared the behavior of the nonphased ribosome-mRNA complex, (-)tRNA(Asp), to the one of the phased complex, (+)tRNA(Asp), in the absence and in the presence of eRF1, the eukaryotic class 1 translation termination factor of human origin. The patterns of cross-links obtained for the three complexes were similar to those previously reported for rabbit ribosomes [Chavatte, L., et al. (2001) Eur. J. Biochem. 268, 2896-2904]. Cross-links involving proteins S2, S3, S3a, and S30 were poorly dependent on the presence of tRNA(Asp) and eRF1. Cross-linking to nucleotide C1696 of 18S rRNA occurred in all complexes, but its yield was at least two times higher in the phased complex with an empty A site than in the nonphased complex or when the A site was occupied by eRF1. In contrast, protein S15 cross-linked only in the phased complex in the absence of eRF1. The data obtained point to notable differences in organization of the decoding site between mammalian and prokaryotic ribosomes and to large internal mobility of the components of the tRNA (eRF1)-free A site.

Base Sequence↗

PubMed2003

Stop codons and UGG promote efficient binding of the polypeptide release factor eRF1 to the ribosomal A site.

To investigate the codon dependence of human eRF1 binding to the mRNA-ribosome complex, we examined the formation of photocrosslinks between ribosomal components and mRNAs bearing a photoactivable 4-thiouridine probe in the first position of the codon located in the A site. Addition of eRF1 to the phased mRNA-ribosome complexes triggers a codon-dependent quenching of crosslink formation. The concentration of eRF1 triggering half quenching ranges from low for the three stop codons, to intermediate for s4UGG and high for other near-cognate triplets. A theoretical analysis of the photochemical processes occurring in a two-state bimolecular model raises a number of stringent conditions, fulfilled by the system studied here, and shows that in any case sound KD values can be extracted if the ratio mT/KD<<1 (mT is total concentration of mRNA added). Considering the KD values obtained for the stop, s4UGG and sense codons (approximately 0.06 microM, 0.45 microM and 2.3 microM, respectively) and our previous finding that only the stop and s4UGG codons are able to promote formation of an eRF1-mRNA crosslink, implying a role for the NIKS loop at the tip of the N domain, we propose a two-step model for eRF1 binding to the A site: a codon-independent bimolecular step is followed by an isomerisation step observed solely with stop and s4UGG codons. Full recognition of the stop codons by the N domain of eRF1 triggers a rearrangement of bound eRF1 from an open to a closed conformation, allowing the universally conserved GGQ loop at the tip of the M domain to come into close proximity of the peptidyl transferase center of the ribosome. UGG is expected to behave as a cryptic stop codon, which, owing to imperfect eRF1-codon recognition, does not allow full reorientation of the M domain of eRF1. As far as the physical steps of eRF1 binding to the ribosome are considered, they appear to closely mimic the behaviour of the tRNA/EF-Tu/GTP complex, but clearly eRF1 is endowed with a greater conformational flexibility than tRNA.

Base Sequence↗

PubMed2003

Termination of translation: interplay of mRNA, rRNAs and release factors?

Termination of translation in eukaryotes has focused recently on functional anatomy of polypeptide chain release factor, eRF1, by using a variety of different approaches. The tight correlation between the domain structure and different functions of eRF1 has been revealed. Independently, the role of prokaryotic RF1/2 in GTPase activity of RF3 has been deciphered, as well as RF3 function itself.

Amino Acid Sequence↗

PubMed2002

Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1.

In eukaryotic ribosomes, termination of translation is triggered by class 1 polypeptide release factor, eRF1. In organisms with a universal code, eRF1 responds to three stop codons, whereas, in ciliates with variant codes, only one or two codon(s) remain(s) as stop signals. By mutagenesis of the Y-C-F minidomain of the N domain, we converted an omnipotent human eRF1 recognizing all three stop codons into a unipotent 'ciliate-like' UGA-only eRF1. The conserved Cys127 located in the Y-C-F minidomain plays a critical role in stop codon recognition. The UGA-only response has also been achieved by concomitant substitutions of four other amino acids located at the Y-C-F and NIKS minidomains of eRF1. We suggest that for eRF1 the stop codon decoding is of a non-linear (non-protein-anticodon) type and explores a combination of positive and negative determinants. We assume that stop codon recognition is profoundly different by eukaryotic and prokaryotic class 1 RFs.

Amino Acid Sequence↗

PubMed2002

Omnipotent decoding potential resides in eukaryotic translation termination factor eRF1 of variant-code organisms and is modulated by the interactions of amino acid sequences within domain 1.

In eukaryotes, a single translational release factor, eRF1, deciphers three stop codons, although its decoding mechanism remains puzzling. In the ciliate Tetrahymena thermophila, UAA and UAG codons are reassigned to Gln codons. A yeast eRF1-domain swap containing Tetrahymena domain 1 responded only to UGA in vitro and failed to complement a defect in yeast eRF1 in vivo at 37 degrees C. This finding demonstrates that decoding specificity of eRF1 from variant code organisms resides at domain 1. However, the wild-type eRF1 hybrid fully restored the growth of eRF1-deficient yeast at 30 degrees C. Tetrahymena eRF1 contains a variant sequence, KATNIKD, at the tip of domain 1. The TASNIKD variant of hybrid eRF1 rendered the eRF1-nullified yeast viable, although in an in vitro assay, the same hybrid eRF1 responded only to UGA. Nevertheless, the yeast eRF1 bearing the KATNIKD motif instead of the TASNIKS heptapeptide present in higher eukaryotes remains omnipotent in vivo. Collectively, these data suggest that variant genetic code organisms like Tetrahymena have an intrinsic potential to decode three stop codons in vivo, and that interaction within domain 1 between the KAT tripeptide and other sequences modulates the decoding specificity of Tetrahymena eRF1.

Amino Acid Sequence↗

PubMed2002

Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1.

Class-1 polypeptide chain release factors (RFs) play a key role in translation termination. Eukaryotic (eRF1) and archaeal class-1 RFs possess a highly conserved Asn-Ile-Lys-Ser (NIKS) tetrapeptide located at the N-terminal domain of human eRF1. In the three-dimensional structure, NIKS forms a loop between helices. The universal occurrence and exposed nature of this motif provoke the appearance of hypotheses postulating an essential role of this tetrapeptide in stop codon recognition and ribosome binding. To approach this problem experimentally, site-directed mutagenesis of the NIKS (positions 61-64) in human eRF1 and adjacent amino acids has been applied followed by determination of release activity and ribosome-binding capacity of mutants. Substitutions of Asn61 and Ile62 residues of the NIKS cause a decrease in the ability of eRF1 mutants to promote termination reaction in vitro, but to a different extent depending on the stop codon specificity, position, and nature of the substituting residues. This observation points to a possibility that Asn-Ile dipeptide modulates the specific recognition of the stop codons by eRF1. Some replacements at positions 60, 63, and 64 cause a negligible (if any) effect in contrast to what has been deduced from some current hypotheses predicting the structure of the termination codon recognition site in eRF1. Reduction in ribosome binding revealed for Ile62, Ser64, Arg65, and Arg68 mutants argues in favor of the essential role played by the right part of the NIKS loop in interaction with the ribosome, most probably with ribosomal RNA.

Amino Acid Sequence↗