Interaction of nascent chains with the ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae.
As translation proceeds, nascent polypeptides pass through an exit tunnel that traverses the large ribosomal subunit. Three ribosomal proteins, termed Rpl4, Rpl17, and Rpl39 expose domains to the interior of the exit tunnel of eukaryotic ribosomes. Here we generated ribosome-bound nascent chains in a homologous yeast translation system to analyze contacts between the tunnel proteins and nascent chains. As model proteins we employed Dap2, which contains a hydrophobic signal anchor (SA) segment, and the chimera Dap2α, in which the SA was replaced with a hydrophilic segment, with the propensity to form an α-helix. Employing a newly developed FLAG exposure assay, we find that the nascent SA segment but not the hydrophilic segment adopted a stable, α-helical structure within the tunnel when the most C-terminal SA residue was separated by 14 residues from the peptidyl transferase center. Using UV cross-linking, antibodies specifically recognizing Rpl17 or Rpl39, and a His6-tagged version of Rpl4, we established that all three tunnel proteins of yeast contact the SA, whereas only Rpl4 and Rpl39 also contact the hydrophilic segment. Consistent with the localization of the tunnel exposed domains of Rpl17 and Rpl39, the SA was in contact with Rpl17 in the middle region and with Rpl39 in the exit region of the tunnel. In contrast, Rpl4 was in contact with nascent chain residues throughout the ribosomal tunnel.
Institute of Biochemistry and Molecular Biology, ZBMZ Centre for Biochemistry and Molecular Cell Research, University of Freiburg, Stefan-Meier-Strasse 17, D-79104 Freiburg, Germany; Centre for Biological Signalling Studies (BIOSS), University of Freiburg, D-79104 Freiburg, Germany.
Ying
Y
Zhang
Institute of Biochemistry and Molecular Biology, ZBMZ Centre for Biochemistry and Molecular Cell Research, University of Freiburg, Stefan-Meier-Strasse 17, D-79104 Freiburg, Germany.
Tina
T
Wölfle
Institute of Biochemistry and Molecular Biology, ZBMZ Centre for Biochemistry and Molecular Cell Research, University of Freiburg, Stefan-Meier-Strasse 17, D-79104 Freiburg, Germany; Centre for Biological Signalling Studies (BIOSS), University of Freiburg, D-79104 Freiburg, Germany. Electronic address: sabine.rospert@biochemie.uni-freiburg.de.
Sabine
S
Rospert
D010458:Peptidyl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000502:physiology*; D017433:Protein Structure, Secondary; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C045547:RPL39 protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D010458:Peptidyl Transferases
Newly Synthesized Polypeptides; Protein Folding; Protein Synthesis; Ribosomes; Site-specific Cross-linking; Translation; Yeast