The cap structure in eukaryotic messenger RNA as a mark of a strand carrying protein information

Miura, K.

Advances in Biophysics 14: 205-238


ISSN/ISBN: 0065-227X
PMID: 7015810
Document Number: 3298
To clarify the specific structure of the strand carrying genetic information in the duplex of genome nucleic acid, the structure of the genome and its transcript were studied on double-stranded RNA containing a virus, especially silkworm cytoplasmic polyhedrosis virus. In these virus, the genome is divided into some 10 segments. It was found that one strand of the double helix in each segment was methylated at the ribose moiety of the 5'-terminal nucleotide, showing a clear difference between the two strands. The virus carrying double-stranded RNA contains RNA ploymerase in its coat, and it can synthesize messenger RNA (mRNA) in vitro. Analysis of the terminal structure of a genome transcript showed that the same strand as the 5' -methylated strand in the genome duplex was copied as an mRNA. If the methyl donor S-adenosylmethionine was added to the mRNa-synthesizing system in vitro, the transcript mRNA was methylated at the 5'terminus; furthermore, it was discovered that the 5'-terminal phosphates were blocked by 7-methylguanosine to become m 7G 5'pppAm-G-. One strand of the genome double helix was also blocked by 7-methylguanosine at the 5'-terminus. Therefore, in cytoplasmic polyhedrosis virus, the strand carrying protein information has a 7-methylguanylic acid blocking the structure at the 5'-terminus. Following after our discovery, a similar structure has been reported in many eukaryotic viral RNAs and eukaryotic, either cellular or viral, mRNAs, and it came to be generally called the 5'"cap" structure of mRNA. The 5'-cap structure was not found in bacterial mRNA. Except for mRNAs in a eukaryotic cell, the cap structure has been detected only in nuclear 5.7S RNA and 5.9S RNA, which carry the 2, 2, 7-trimethylguanosine (rni,2,7G)-cap at the 5'-terminal. The conformation of the 5'-cap region was studied by ultraviolet, circular dichroism, and fluorescent spectra with the use of a chemically-synthesized cap structure. The cap has a base-stacked structure at pH 7. Under optimal conditions for protein synthesis, between pH 7 and 8, the cap structure shows a drastic change in its conformation, when it is a reactive state. Protein synthesis in a eukaryotic system is inhibited by the addition of a cap structure or 7-methylguanylic acid. Strong inhibition is specifically caused by 7-methylguanylic acid among the methylated analogues of guanylic acid. Initiation complex formation in protein synthesis is also inhibited by the addition of 7-methylguanylic acid. If the 7-methylguanylic acid residue in the cap of mRNA was deleted specifically by tobacco pyrophosphatase, mRNA lost its ability to synthesize protein and to form the initiation complex. However, the inhibition of protein synthesis by the addition of 7-methvlguanylic acid and the loss of protein-synthesizing ability by deletion of the cap were not perfect, so that the cap structure may function cooperatively with the initiation codon, the higher order structure near the 5'-terminus of mRNA, and the sequence complementary to the ribosomal RNA to form the initiation complex efficiently. The cap structure is effective for stabilization of mRNA.

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The cap structure in eukaryotic messenger RNA as a mark of a strand carrying protein information