An important 2'-OH group for an RNA-protein interaction

Abstract

We have investigated the role of 2'-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNA(Cys) and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2'-OH by 2'-deoxy or 2'-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2'-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2'-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2'-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2'-OH group instead provides a monitor of the steric environment during the RNA-synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2'-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

Figure 1

(A) Sequence and cloverleaf structure of E.coli tRNA^Cys, where the boxed U73 is the major determinant for aminoacylation with cysteine. (B) Sequence and secondary structure of the synthetic E.coli microhelix^Cys, where U73 is boxed. (C) Sequence and secondary structure of the synthetic M.pneumoniae microhelix^Cys, where U73 is boxed. (D) Sequence and cloverleaf structure of B.subtilis tRNA^Cys. The arrow indicates separation of the 5′- and 3′-fragments for reconstitution.

Figure 2

Chemical structures and features of the (A) 2′-OH, (B) 2′-H, (C) 2′-O-methyl and (D) 2′-O-propyl moieties on uridine.

Figure 3

Imino spectrum at 15°C of the M.pneumoniae microhelix tRNA^Cys. The assignments are indicated above their corresponding resonance position. The imino proton resonances for U8 and G11 confirm that a UUCG tetraloop is formed. The presence of an imino peak for G1 is exceptional at this high a temperature and supports the structural model wherein stacking of A76 onto G1 serves to protect the G1 imino proton from exchange.

Figure 4

Selected regions of the 250 ms mixing time NOESY spectrum acquired at 15°C of the M.pneumoniae microhelix tRNA^Cys. These sections show the NOE connectivities between the nucleotides of the closing stem base pair and the UCCA tail.

Figure 5

The NMR-based fold-back model of the UCCA end in the M.pneumoniae microhelix^Cys, which shows the bases of C71, C72, U73, C74, C75, A76, G1 and G2 as planes and 2′-OH groups as large balls. The 2′-OH group of U73 is colored orange whereas those of all others are in black.