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NMR solution structure and function of the C-terminal domain of eukaryotic class 1 polypeptide chain release factor.

Mantsyzov AB, Ivanova EV, Birdsall B, Alkalaeva EZ, Kryuchkova PN, Kelly G, Frolova LY, Polshakov VI - FEBS J. (2010)

Bottom Line: The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure.Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor.The results provide new insights into the possible role of the C-domain in the process of translation termination.

View Article: PubMed Central - PubMed

Affiliation: Center for Magnetic Tomography and Spectroscopy, M. V. Lomonosov Moscow State University, Russia.

ABSTRACT
Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three-domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C-terminal domain (C-domain). The high-resolution NMR structure of the human C-domain (residues 277-437) has been determined in solution. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329-372), which was ill-defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using (15)N-relaxation experiments, showed that the C-terminal tail 414-437 and the minidomain are the most flexible parts of the human C-domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C-domain onto the available crystal structure of full-length human eRF1 shows that the minidomain is close to the stop codon-recognizing N-terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C-domain in the process of translation termination.

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The rate of peptidyl-tRNA hydrolysis in response to human eRF1 with mutations in the minidomain. The 35S-labeled tetrapeptide (MVHL) released as a function of time from termination complexes formed with UAA (A), UAG (B) and UGA (C) stop codons by wild-type eRF1 (solid circles) or mutant forms of eRF1 is shown. The background release of tetrapeptide in the absence of eRF1 was subtracted from all graphs. The data are normalized to the release given by wild-type eRF1 at 15 min.
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fig07: The rate of peptidyl-tRNA hydrolysis in response to human eRF1 with mutations in the minidomain. The 35S-labeled tetrapeptide (MVHL) released as a function of time from termination complexes formed with UAA (A), UAG (B) and UGA (C) stop codons by wild-type eRF1 (solid circles) or mutant forms of eRF1 is shown. The background release of tetrapeptide in the absence of eRF1 was subtracted from all graphs. The data are normalized to the release given by wild-type eRF1 at 15 min.

Mentions: Superposition of the NMR structure of the human C-domain on the full-length crystal structure of eRF1 reveals that the minidomain is located close to or adjacent to the N-domain (see Discussion), which is responsible for the stop codon recognition (Fig. 6). One can assume that complex dynamic behavior of the minidomain may influence the state of the N-domain and may therefore modify the efficiency of the decoding process. To verify this hypothesis, we generated a series of mutant forms of eRF1 with the replacement of Tyr331, His334, His356, Phe357, Asp359, Gly363, Glu365, His366 and Glu370 by alanine. These point mutants were further assayed in a reconstituted in vitro eukaryotic translation system containing 60S and 40S ribosomal subunits, mRNA with different stop codons, aminoacylated tRNAs, and individual purified translation factors [13]. The efficiency of termination was estimated from the amount of released 35S-labeled peptide at several time intervals. The mutations Y331A, H356A, F357A, D359A, G363A and E365A in the loop region were found to increase the termination efficiency of the ribosomal complex with the UAG stop codon, whereas the peptide release rate did not change significantly when UAA or UGA stop codons were used (Fig. 7). The maximum impact on the peptidyl-tRNA hydrolysis was found for the E365A and D359A mutants, in which negatively charged residues were replaced by alanine. One can speculate that the negative charges reduce the efficiency of the minidomain interaction with mRNA. It is also worth noting that the maximum impact was observed for mutations in the flexible loop 357–367. Replacement of His334, His366 and Glu370 did not change the peptide release rate, regardless of the stop codon used (Fig. S7).


NMR solution structure and function of the C-terminal domain of eukaryotic class 1 polypeptide chain release factor.

Mantsyzov AB, Ivanova EV, Birdsall B, Alkalaeva EZ, Kryuchkova PN, Kelly G, Frolova LY, Polshakov VI - FEBS J. (2010)

The rate of peptidyl-tRNA hydrolysis in response to human eRF1 with mutations in the minidomain. The 35S-labeled tetrapeptide (MVHL) released as a function of time from termination complexes formed with UAA (A), UAG (B) and UGA (C) stop codons by wild-type eRF1 (solid circles) or mutant forms of eRF1 is shown. The background release of tetrapeptide in the absence of eRF1 was subtracted from all graphs. The data are normalized to the release given by wild-type eRF1 at 15 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984548&req=5

fig07: The rate of peptidyl-tRNA hydrolysis in response to human eRF1 with mutations in the minidomain. The 35S-labeled tetrapeptide (MVHL) released as a function of time from termination complexes formed with UAA (A), UAG (B) and UGA (C) stop codons by wild-type eRF1 (solid circles) or mutant forms of eRF1 is shown. The background release of tetrapeptide in the absence of eRF1 was subtracted from all graphs. The data are normalized to the release given by wild-type eRF1 at 15 min.
Mentions: Superposition of the NMR structure of the human C-domain on the full-length crystal structure of eRF1 reveals that the minidomain is located close to or adjacent to the N-domain (see Discussion), which is responsible for the stop codon recognition (Fig. 6). One can assume that complex dynamic behavior of the minidomain may influence the state of the N-domain and may therefore modify the efficiency of the decoding process. To verify this hypothesis, we generated a series of mutant forms of eRF1 with the replacement of Tyr331, His334, His356, Phe357, Asp359, Gly363, Glu365, His366 and Glu370 by alanine. These point mutants were further assayed in a reconstituted in vitro eukaryotic translation system containing 60S and 40S ribosomal subunits, mRNA with different stop codons, aminoacylated tRNAs, and individual purified translation factors [13]. The efficiency of termination was estimated from the amount of released 35S-labeled peptide at several time intervals. The mutations Y331A, H356A, F357A, D359A, G363A and E365A in the loop region were found to increase the termination efficiency of the ribosomal complex with the UAG stop codon, whereas the peptide release rate did not change significantly when UAA or UGA stop codons were used (Fig. 7). The maximum impact on the peptidyl-tRNA hydrolysis was found for the E365A and D359A mutants, in which negatively charged residues were replaced by alanine. One can speculate that the negative charges reduce the efficiency of the minidomain interaction with mRNA. It is also worth noting that the maximum impact was observed for mutations in the flexible loop 357–367. Replacement of His334, His366 and Glu370 did not change the peptide release rate, regardless of the stop codon used (Fig. S7).

Bottom Line: The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure.Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor.The results provide new insights into the possible role of the C-domain in the process of translation termination.

View Article: PubMed Central - PubMed

Affiliation: Center for Magnetic Tomography and Spectroscopy, M. V. Lomonosov Moscow State University, Russia.

ABSTRACT
Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three-domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C-terminal domain (C-domain). The high-resolution NMR structure of the human C-domain (residues 277-437) has been determined in solution. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329-372), which was ill-defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using (15)N-relaxation experiments, showed that the C-terminal tail 414-437 and the minidomain are the most flexible parts of the human C-domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C-domain onto the available crystal structure of full-length human eRF1 shows that the minidomain is close to the stop codon-recognizing N-terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C-domain in the process of translation termination.

Show MeSH
Related in: MedlinePlus