Analysis of Escherichia coli 4.5S RNA binding affinity to Ffh and EF-G.
Escherichia coli 4.5S RNA is a member of the signal recognition particle RNA family that binds to Ffh and EF-G proteins in vivo. To assess the binding affinity of E. coli 4.5S RNA, wild-type Ffh and a series of amino terminal truncated EF-G mutants with a histidine tag were over-expressed in Escherichia coli and purified. Among them, EF-G mutants with a deletion of all upstream sequences up to and including the second or the third GTP binding sequence element were expressed at high levels and bound with the same activity as wild-type EF-G. Nitrocellulose filter binding assays revealed that the binding affinity values (M(1/2)) for Ffh and EF-G, defined as the concentration giving half-maximal binding, were 0.15 microM and 1.5 microM, respectively. Moreover, we also show that very little EF-G can form a stable complex with 4.5S RNA in vivo, whereas almost all Ffh binds to 4.5S RNA.
Suzuma
S
S
Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki, Japan.
Hayashi
K
K
Nakamura
K
K
Yamane
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine; D008969:Molecular Sequence Data; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D018271:Signal Recognition Particle / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D020653:Peptide Elongation Factor G; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D018271:Signal Recognition Particle; D006639:Histidine