Binding of Escherichia coli initiation factor IF2 to 30S ribosomal subunits: a functional role for the N-terminus of the factor.
In the initiation step of bacterial protein synthesis initiation factor IF2 has to join the 30S ribosomal subunit in order to promote the binding of the fMet-tRNAMetf. In order to identify regions within IF2 which may be involved in the primary ribosome-factor interaction, we have constructed several C-terminal and N-terminal truncated forms of the factor as well as isolated structural domains, and tested them in a 30S ribosomal binding assay in vitro. Monoclonal antibodies with epitopes located within the two N-terminal domains of IF2 were used in these experiments. Hitherto, no function has been allocated to the N-terminal region of IF2. Here we show that a mutant consisting of the two N-terminal domains has intrinsic affinity to the ribosomal subunit. Furthermore, a deletion mutant of IF2 which is lacking the two N-terminal domains shows negligible affinity. Moreover mAb with epitopes located within domain II strongly inhibits the binding capacity of IF2 to the 30S ribosomal subunit, whereas mAb with epitopes mapped within domain I do not affect the binding of the factor. The C-terminal domain of IF2 shows no affinity for the small ribosomal subunit. In addition, mutants with C-terminal deletions are not significantly affected in this interaction. Therefore, we conclude that the N-terminus of IF2 has affinity per se to bind the ribosomal subunit, with domain II being directly involved in the interaction.
Moreno
J M
JM
Department of Biostructural Chemistry, Institute of Molecular and Structural Biology, Aarhus University, Gustav Wieds Vej 10, Aarhus C, DK-8000, Denmark.
Kildsgaard
J
J
Siwanowicz
I
I
Mortensen
K K
KK
Sperling-Petersen
H U
HU
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039881:Prokaryotic Initiation Factor-2; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012269:Ribosomal Proteins; C040469:ribosomal protein S30