Studies on the Sequence of the 3′‐Terminal Region of Turnip‐Yellow‐Mosaic‐Virus RNA

Abstract

A fragment representing the 3′‐terminal ‘tRNA‐like’ region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli‘RNase P’. This fragment, which is 112 ± 3‐nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P‐end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G‐U‐U‐C‐R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5′ end and 16 nucleotides from the 3′ end of this fragment has been deduced. The nucleotide sequence at the 5′ end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.

Chemical reactivity ofE. coli5S RNAin situin the 50S ribosomal subunit

“…Using in vitro labeling methods (24,25) we have identified the two A residues of E. coli 5S RNA which are most accessible in the 50S ribosomal subunit to chemical modification by monoperphthalic acid as A73 and Ag9. These two A residues are not the tandem As present within the sequence C-G-A-A46-, which has been postulated to interact with the constant T-Y-C-G-sequence of tRNAs during the binding of aminoacyl-tRNAs to the ribosomal A site, but are among those nucleotides which are strictly conserved in all prokaryotic 5S RNAs (3,28,33).…”

“…1 A-D) are indicated in Table III. Identification of sequences was based mostly on their location on the the fingerprint (7) and 5'-end group analyses; several of these identifications were also confirmed by partial digestion with snake venom phophodiesterase followed by analysis by twodimensional homochromatography (25). The two oligonucleotides whose yield decreases with time of exposure to monoperphthalic acid are 13 (from 1.2mole to 0.18mole) and 18 (from 0.87mole to …”

“…(d) Sequence analysis of 5S RNA and identification of N-oxidized adenines in 5S RNA 5S RNA isolated from untreated and monoperphthalic acid treated 50S ribosomal subunits were digested with pancreatic RNase and the oligonucleotides present in such digests were labeled with 32P at their 5'-end as previously described (24,25). The 5'-(32p)-labeled oligonucleotides were separated by fingerprinting (26) and their sequences were established by partial digestion with snake venom phosphodiesterase followed by analysis of the partial digests by two dimensional homochromatography (25,27). RESULTS (a) Chemical modification and biological activity of 5S RNA E. coli 50S ribosomal subunits were treated with monoperphthalic acid under the conditions outlined in Materials and Methods.…”

“…(c) Equilibrium dialysis Synthesis of 3H labeled U-U-C-G, equilibrium dialysis experiments and calculations of association constants were carried out as published (12,22,23). (d) Sequence analysis of 5S RNA and identification of N-oxidized adenines in 5S RNA 5S RNA isolated from untreated and monoperphthalic acid treated 50S ribosomal subunits were digested with pancreatic RNase and the oligonucleotides present in such digests were labeled with 32P at their 5'-end as previously described (24,25). The 5'-(32p)-labeled oligonucleotides were separated by fingerprinting (26) and their sequences were established by partial digestion with snake venom phosphodiesterase followed by analysis of the partial digests by two dimensional homochromatography (25,27).…”

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

“…The known sequence at the 3' end of both TYMV RNAs (7,8) can base-pair with the 5'-terminal sequence in the vicinity of the potential initiation codon. The costs of publication of this article were defrayed in part by the payment of page charges.…”