Two Homologous EF-G Proteins from Pseudomonas aeruginosa Exhibit Distinct Functions

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• Alternative Title:
  PLoS One
• Personal Author:
  Palmer, Stephanie O. ; Rangel, Edna Y. ; Hu, Yanmei ; Tran, Alexis T. ; Bullard, James M.
• Description:
  Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k cat) for the hydrolysis of GTP than EF-G1A; 0.2 s(-1) vs. 0.04 s(-1). These values resulted in specificity constants (k cat (obs)/K M) for EF-G1A and EF-G1B of 0.5 x 10(3) s(-1) M(-1) and 3.0 x 10(3) s(-1) M(-1), respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.
• Subjects:
  Bacterial Proteins Fusidic Acid GTP Phosphohydrolases Guanosine Triphosphate Hydrolysis Kinetics Peptide Elongation Factor G Poly U Pseudomonas Aeruginosa Ribosomes Sequence Analysis

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• Source:
  PLoS One. 2013; 8(11).
• Pubmed ID:
  24260360
• Pubmed Central ID:
  PMC3832671
• Document Type:
  Journal Article
• Funding:
  1SC3GM098173-01A1/GM/NIGMS NIH HHS/United States ; 75DP001812/DP/NCCDPHP CDC HHS/United States ; SC3 GM098173/GM/NIGMS NIH HHS/United States
• Volume:
  8
• Issue:
  11
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:87b1b0d3e4584ac0e6003e4652bb36ecb5ab2a85ed5dcd490607ef7137e622f3
• Download URL:
  https://stacks.cdc.gov/view/cdc/22555/cdc_22555_DS1.pdf
• File Type:
  [PDF - 467.52 KB ]