Ribosome binding to inosine-substituted mRNAs in the absence of ATP and mRNA factors.
Incubating ribosomes and eukaryotic initiation factor eIF3 with an inosine-substituted mRNA (where the mRNA secondary structure is strongly reduced) in the absence of ATP and other protein synthesis factors produces a 40 S ribosome.mRNA complex. When Met-tRNAMeti and eIF2 are added, a 60 S ribosome subunit attaches forming an 80 S ribosome.mRNA complex. ATP and the three mRNA factors, eIF4B, cap-site factor, and eIF4A, strongly stimulate the attachment of the 60 S subunit. In the absence of Met-tRNAMeti, the 60-S subunit does not attach, and adding ATP and the mRNA factors inhibits the accumulation of 40 S ribosome.inosine mRNA complexes. These results indicate that a 40 S ribosome, probably in a complex with eIF3, has an intrinsic capacity to attach to mRNA. Further, they suggest that Met-tRNAMeti may interact in a subsequent step to stabilize the 40 S ribosome.mRNA complex and allow the attachment of a 60 S ribosome subunit. Although seen most clearly with the inosine-substituted mRNAs, the 40 S ribosome reaction is also obtained with "guanosine" mRNA. A 40 S ribosome attaches to guanosine mRNA without ATP and mRNA factors when an incubation mixture containing ribosomes, eIF3, and mRNA is fixed with glutaraldehyde. In addition, a 40 S ribosome.guanosine mRNA complex can be obtained without glutaraldehyde in incubations containing ATP and the three mRNA factors in the absence of Met-tRNAMeti. The latter reaction is limited because of the instability of the 40 S ribosome.mRNA complex in the absence of Met-tRNA. Nevertheless, its authenticity is indicated by its full dependence upon ATP and the three mRNA factors. The lack of factor requirement for the formation of 40 S ribosome complexes with inosine-substituted mRNAs indicates that ATP and the three mRNA factors function primarily to unwind the secondary structure of a guanosine mRNA. Data relevant to a role for ATP in facilitating ribosome migration on an mRNA are also discussed.
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Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
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Marcus
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D007288:Inosine; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013937:Thymidine Kinase / Q000235:genetics
D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D012345:RNA, Transfer, Amino Acid-Specific; D012358:RNA, Transfer, Met; D012367:RNA, Viral; D007288:Inosine; D000255:Adenosine Triphosphate; D013937:Thymidine Kinase