Nucleotide sequence of 5' terminus of alfalfa mosaic virus RNA 4 leading into coat protein cistron.

Abstract

The sequence of the 5'-terminal 74 nucleotides of alfalfa mosaic virus RNA 4, the mRNA for the viral coat protein, has been deduced by using various new techniques for abe ing the RNA at the 5' end with 32P and for sequencing the 5'-32P-labeled RNA. The sequence is NpppGUUUUUAUUU-UUAAUUUUCUUUCAAAUACUUCCAUCAUGAGUUCUUC-ACAAAAGAAAGCUGGUGGGAAAGCUGG. The AUG initiator codon is located 36 nucleotides in from the 5' end; the nucleotide sequence beyond corresponds to the amino acid sequence of the coat rotein. This 5' noncoding region is rich in U (58% U); except for the 5'-terminal G, the next G in is part of the initiator AUG codon. "Cap" structures of the type m7G(5')ppp(5')N are found at the 5' terminus of many eukaryotic mRNAs and have been implicated in ribosome binding (1). The detection of other possible common signals has been hampered by the absence, until recently, of comparative sequence data on the 5'-noncoding regions of these molecules. For some time, only the sequence of the nine nucleotides between the "cap" and the AUG initiator codon in brome mosaic virus (BMV) RNA 4 was known (2). Recently, nucleotide sequences preceding the initiator codon have been published for rabbit a-and O-globin mRNA (3-5), the simian virus 40 VP1 gene (6), oncornaviral RNA (7-9), reovirus mRNA (10), vesicular stomatitis virus mRNA (11), and tobacco mosaic virus RNA (12). Comparison of these data reveals no readily identifiable recognition signals and shows that the distance between the "cap" and the first AUG codon is variable. The synthesis of these RNA species involves various different enzymes and templates, which may be either DNA or RNA, and the sequence at their 5' end may be determined by constraints other than ribosome recognition alone.We have analyzed the sequence of the 5'-noncoding region in RNA 4 of alfalfa mosaic virus (AlMV), which is functionally equivalent to BMV-RNA 4. AIMV, like BMV, has a genome consisting of three RNA molecules, the coat protein cistron being located in RNA 3, the smallest genome fragment (13 making up the BMV genome are infectious as such, the AIMV genome has to be supplemented with a small amount of coat protein, or the RNA 4, which presumably acts as the coat protein messenger (19). Moreover, the overall sequence homology between the genomes of the two viruses is negligible (20).In the course of this study, we made use of a number of novel rapid sequencing techniques, including a gel sequencing method similar in principle to the DNA sequencing method of Maxam and Gilbert (21) and a "wandering spot" analysis that uses two-dimensional polyacrylamide gels (details to be published elsewhere). All the methods involve first the in vitro enzymatic labeling of the 5' terminus of AIMV RNA 4, after removal of the "cap" by periodate oxidation, #-elimination, and phosphomonoesterase digestion (3). The results enable us to deduce the sequence of the 5'-terminal 74 nucleotides of AIMV RNA 4. The AUG initiator codon of the coat protein cistron was found to be preceded by 36 nucleotides. The ...

Capped mRNAs with Reduced Secondary Structure Can Function in Extracts from Poliovirus-Infected Cells

“…To further test our model, we analyzed the translation of the capped alfalfa mosaic virus 4 (AMV-4) RNA, which contains an adenosineuracil-rich 5' leader region (18) and hence cannot form a stable secondary structure, as predicted by computer-aided analysis (P. Auron, personal communication). Consequently, although this mRNA is capped, we might expect its translation to be less dependent on the cap structure.…”

3'-Terminal nucleotide sequence of alfalfa mosaic virus RNA 4.

“…The relative positions of guanine, adenine, and, by difference, pyrimidine residues were determined on onedimensional sequencing gels as described (1,17 3 ,ug of carrier in 20 Ml of 50 mM NaHCO3 (pH 9.0) for 7 min at 1001C. All enzyme incubations (control without enzyme included) were at 50'C for 15 min, as described by Donis-Keller et al (17).…”

“…3 by the letter Y, was established by two-dimensional sequencing gels (1,18). Partial alkaline digests of the 5'-labeled fragment were electrophoresed in a 7 M urea/10% polyacrylamide gel at pH 3.5, and 16-cm-long strips were excised.…”

“…Recovery of the labeled fragments was by the method of De Wachter and Fiers (16). Sequencing techniques were essentially as described (1,17). It appeared necessary to stir the urea solutions for a few minutes with an ion exchanger (Rexyn I-300) before use to avoid streaking, especially upon gel electrophoresis at pH 3.5. of which comigrated with 29B.…”

Nucleotide sequence at the 5′ extremity of turnip yellow mosaic virus genome RNA

“…The best candidate for a functional initiation codon in the 5'-terminal sequence is 94 nucleotides from the 5' end. The region preceding this possible initiation codon has little in common with the 5' noncoding regions of TYMV coat protein mRNA (unpublished data) or any other plant viral RNAs characterized to date (3)(4)(5) but does bear a striking resemblance to the 5' extremity of rabbit ,3-globin mRNA (6).…”