Interaction of protein synthesis initiation factors with the mRNA cap structure.
The mechanism of mRNA recognition by proteins interacting with the mRNA cap structure was investigated by photochemical cross-linking of proteins with 32P-labelled reoviral RNAs. Using ribosomal washes as a source of eukaryotic protein synthesis initiation factors, we identified the well-known cap binding proteins eIF-4B and -4E, but eIF-2 and eIF-3 as well. The interplay of purified eIF-4A, -4B, and -4F was studied in relation to ATP dependence and cap analogue sensitivity of cap binding. Next to their well-known roles in the initiation process, eIF-2 and eIF-3 also cross-linked to the 5' cap. eIF-2 stimulated eIF-4B and -4E cross-linking, an observation that has been previously described more extensively. The interaction of eIF-2 with the 5' end of mRNA was extremely sensitive to K(+)-ions and was resistant to a high concentration of Mg(2+)-ions; this influence of mono- and divalent ions was in contrast with the cross-linking of eIF-4B and -4E. Optimal interaction of these factors was obtained at moderate K+ concentration and low Mg(2+)-ion concentrations. eIF-2 cross-linking was sensitive to high protein to mRNA ratios indicating a weak affinity as compared to eIF-4E and -4B. The interaction of eIF-3 with the cap of mRNA is also weak as it was counteracted by all other cap binding proteins, leading to an inability to detect the cross-linking of this protein in crude eIF preparations. Time kinetics of formation of complexes suggested eIF-2 to be one of the first factors to interact with mRNA. Preformed RNA-protein complexes were dissociated after cap analogue addition, suggesting reversible interactions between RNA and proteins.
van Heugten
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Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Thomas
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Voorma
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HO
D000255:Adenosine Triphosphate / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000378:metabolism; D039621:Eukaryotic Initiation Factor-3; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D008274:Magnesium / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D012314:RNA Cap Analogs / Q000494:pharmacology; D012315:RNA Caps / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism
D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D012314:RNA Cap Analogs; D012315:RNA Caps; D012367:RNA, Viral; C031799:eIF-4B; D000255:Adenosine Triphosphate; D008274:Magnesium; D011188:Potassium