The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain.
Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.
Helgstrand
Magnus
M
Department of Biophysical Chemistry, Lund University, Box 124, SE-22100 Lund, Sweden.
Mandava
Chandra S
CS
Mulder
Frans A A
FA
Liljas
Anders
A
Sanyal
Suparna
S
Akke
Mikael
M
D017124:Conserved Sequence; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D010454:Peptide Termination Factors / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2 / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012709:Serum Albumin / Q000737:chemistry; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D010454:Peptide Termination Factors; D039881:Prokaryotic Initiation Factor-2; D012269:Ribosomal Proteins; D012709:Serum Albumin; C489007:prfC protein, E coli; C512844:rplL protein, E coli; D010444:Peptide Elongation Factor Tu