Ribosomal protein L7/L12 is required for GTPase translation factors EF-G, RF3, and IF2 to bind in their GTP state to 70S ribosomes.
Ribosomal protein L7/L12 is associated with translation initiation, elongation, and termination by the 70S ribosome. The guanosine 5' triphosphate hydrolase (GTPase) activity of elongation factor G (EF-G) requires the presence of L7/L12, which is critical for ribosomal translocation. Here, we have developed new methods for the complete depletion of L7/L12 from Escherichia coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTPase factors EF-G, RF3, IF2, and LepA. Upon removal of L7/L12 from ribosomes, the GTPase activities of EF-G, RF3, and IF2 decreased to basal levels, while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the GTPase activities of all GTPases returned to full activity. Moreover, ribosome binding assays indicated that EF-G, RF3, and IF2 require L7/L12 for stable binding in the GTP state, and LepA retained > 50% binding. Lastly, an EF-G∆G' truncation mutant possessed ribosome-dependent GTPase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome-dependent GTPases that harbor direct interactions to the L7/L12 C-terminal domains, either through a G' domain (EF-G, RF3) or a unique N-terminal domain (IF2). Furthermore, we hypothesize this interaction is concomitant with counterclockwise ribosomal intersubunit rotation, which is required for translocation, initiation, and post-termination.
Department of Chemistry, Western Washington University, Bellingham, WA, USA.
Markus A
MA
Carlson
Department of Chemistry, Western Washington University, Bellingham, WA, USA.
Bassam G
BG
Haddad
Department of Chemistry, Western Washington University, Bellingham, WA, USA.
Amanda J
AJ
Weis
Department of Chemistry, Western Washington University, Bellingham, WA, USA.
Colby S
CS
Blackwood
Department of Chemistry, Western Washington University, Bellingham, WA, USA.
Catherine D
CD
Shelton
Department of Chemistry, Western Washington University, Bellingham, WA, USA.
Michelle E
ME
Wuerth
Department of Chemistry, Western Washington University, Bellingham, WA, USA.
Justin D
JD
Walter
Department of Chemistry, Western Washington University, Bellingham, WA, USA.
Paul Clint
PC
Spiegel
D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism; D010454:Peptide Termination Factors / Q000378:metabolism*; D039881:Prokaryotic Initiation Factor-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D012270:Ribosomes / Q000378:metabolism*
D029968:Escherichia coli Proteins; C515651:LepA protein, E coli; D020653:Peptide Elongation Factor G; D010448:Peptide Initiation Factors; D010454:Peptide Termination Factors; D039881:Prokaryotic Initiation Factor-2; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C489007:prfC protein, E coli; C512844:rplL protein, E coli; D006160:Guanosine Triphosphate
GTPase; elongation factor G; protein L7/L12; ribosome; translation