Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency.
Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.
Berben-Bloemheuvel
G
G
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Kasperaitis
M A
MA
van Heugten
H
H
Thomas
A A
AA
van Steeg
H
H
Voorma
H O
HO
D001483:Base Sequence; D003432:Cross-Linking Reagents; D015967:Gene Expression Regulation, Viral; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics*
D003432:Cross-Linking Reagents; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral