Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acids involved in its ribosomal binding and recycling

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 ≥ to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.

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Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20

Journal of Molecular Biology ◽

10.1016/0022-2836(92)90827-7 ◽

1992 ◽

Vol 228 (2) ◽

pp. 366-386 ◽

Cited By ~ 41

Author(s):

P. Lesage ◽

C. Chiaruttini ◽

M. Graffe ◽

J. Dondon ◽

M. Milet ◽

...

Keyword(s):

Escherichia Coli ◽

Secondary Structure ◽

Translation Initiation ◽

Messenger Rna ◽

Rna Secondary Structure ◽

Ribosomal Proteins ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Translational Coupling

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Organization of the Escherichia coli chromosome around the genes for translation initiation factor IF2 (infB) and a transcription termination factor (nusA)

Journal of Molecular Biology ◽

10.1016/s0022-2836(83)80333-7 ◽

1983 ◽

Vol 167 (2) ◽

pp. 227-243 ◽

Cited By ~ 32

Author(s):

J.A. Plumbridge ◽

M. Springer ◽

M.E. Gottesman

Keyword(s):

Escherichia Coli ◽

Translation Initiation ◽

Transcription Termination ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Termination Factor ◽

Transcription Termination Factor

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mRNA Decay during Herpes Simplex Virus (HSV) Infections: Protein-Protein Interactions Involving the HSV Virion Host Shutoff Protein and Translation Factors eIF4H and eIF4A

Journal of Virology ◽

10.1128/jvi.79.15.9651-9664.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 9651-9664 ◽

Cited By ~ 85

Author(s):

Pinghui Feng ◽

David N. Everly ◽

G. Sullivan Read

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Translation Initiation ◽

Mammalian Cells ◽

Translation Initiation Factor ◽

Site Directed Mutagenesis ◽

Initiation Factor ◽

Virion Host Shutoff ◽

Host Shutoff ◽

Simplex Virus

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.

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