ovalbumin and aminomethyltrioxsalen

We used the chemical reagents dimethylsulfate and 4'-aminomethyl-4,5',8-trimethylpsoralen and the enzyme T1 ribonuclease to compare the 5'-end structure of ovalbumin mRNA in situ in purified hen oviduct nuclei and polysomes with that of the isolated mRNA. The qualitative pattern of structure-dependent base modifications and T1 ribonuclease cleavage sites in intranuclear and polysomal ovalbumin mRNAs was found to be nearly identical to those in isolated ovalbumin mRNA. These structural data are consistent with the presence of a trigonal stem-loop structure at the 5'-end of ovalbumin mRNA (hairpin-1) in nuclei and polysomes. Similar results were obtained for a coding region structure (hairpin-3) in intranuclear ovalbumin mRNA. We have recently shown that hairpin-1 positively affects the rate of ovalbumin mRNA translation in vitro and is part of a high affinity binding site for eucaryotic initiation factor-2 (eIF-2). The presence of hairpin-1 in ovalbumin mRNA in both a pretranslation state (nuclei) and active translation state (polysomes) is consistent with its hypothesized biological function as an intracellular initiation signal that facilitates the translation of this mRNA.

Topics: Adenine; Animals; Base Sequence; Binding Sites; Cell Nucleus; Chickens; Codon; Cytidine; Female; Methylation; Molecular Sequence Data; Nucleic Acid Conformation; Ovalbumin; Photochemistry; Polyribosomes; Ribonuclease T1; RNA, Messenger; Trioxsalen

We have described the reaction of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with hen oviduct mRNA and have investigated the specific effects of AMT photoaddition on ovalbumin mRNA which constitutes 60-70% of oviduct mRNA. The photoreaction of AMT with hen oviduct mRNA appeared to occur in two phases - a rapid monoaddition followed by a slower conversion of monoadducts to diadducts (i.e. crosslinking). Both nondenaturing and denaturing gel electrophoresis revealed a photoreaction time dependent increase in ovalbumin mRNA electrophoretic mobility indicating the formation of a progressively more compact molecular structure. Identical analysis of photoreacted rabbit globin mRNA revealed no change in electrophoretic mobility suggesting that AMT was stabilizing the AU rich secondary structure of ovalbumin mRNA but having no similar effect on the relatively GC rich secondary structure of globin mRNA. Ovalbumin-specific DNA primer extension was used to demonstrate the selective and secondary structure specific photoaddition of AMT to uracil bases at the 5' end of ovalbumin mRNA.

Topics: Animals; Base Sequence; Chickens; Cross-Linking Reagents; DNA; Female; Furocoumarins; Kinetics; Molecular Weight; Nucleic Acid Conformation; Ovalbumin; Oviducts; RNA, Messenger; Trioxsalen