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Structural integrity of {alpha}-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability.
- Source :
-
RNA (New York, N.Y.) [RNA] 2011 Apr; Vol. 17 (4), pp. 687-96. Date of Electronic Publication: 2011 Feb 18. - Publication Year :
- 2011
-
Abstract
- Translation initiation factor eIF5B promotes GTP-dependent ribosomal subunit joining in the final step of the translation initiation pathway. The protein resembles a chalice with the α-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, helix H12 was lengthened or shortened by one or two turns. In addition, six consecutive residues in the helix were substituted by Gly to alter the helical rigidity. Whereas the mutations had minimal impacts on the factor's binding to the ribosome and its GTP binding and hydrolysis activities, shortening the helix by six residues impaired the rate of subunit joining in vitro and both this mutation and the Gly substitution mutation lowered the yield of Met-tRNA(i)(Met) bound to 80S complexes formed in the presence of nonhydrolyzable GTP. Thus, these two mutations, which impair yeast cell growth and enhance ribosome leaky scanning in vivo, impair the rate of formation and stability of the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNA(i)(Met) is bound and that helix H12 rigidity is required to stabilize Met-tRNA(i)(Met) binding.
- Subjects :
- Amino Acid Sequence
Amino Acid Substitution
Basic-Leucine Zipper Transcription Factors biosynthesis
Eukaryotic Initiation Factor-2 genetics
Eukaryotic Initiation Factor-2 metabolism
Eukaryotic Initiation Factors genetics
Eukaryotic Initiation Factors metabolism
Glycine chemistry
Glycine genetics
Glycine metabolism
Guanosine Triphosphate metabolism
Molecular Sequence Data
Mutation
Protein Structure, Secondary
RNA, Transfer, Met metabolism
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae growth & development
Saccharomyces cerevisiae metabolism
Saccharomyces cerevisiae Proteins biosynthesis
Saccharomyces cerevisiae Proteins genetics
Saccharomyces cerevisiae Proteins metabolism
Eukaryotic Initiation Factor-2 chemistry
Eukaryotic Initiation Factors chemistry
Ribosomes metabolism
Saccharomyces cerevisiae Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1469-9001
- Volume :
- 17
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- RNA (New York, N.Y.)
- Publication Type :
- Academic Journal
- Accession number :
- 21335519
- Full Text :
- https://doi.org/10.1261/rna.2412511