Effect of G-1 on histidine tRNA microhelix conformation

Abstract

Histidine tRNAs (tRNA(His)) are unique in that they possess an extra 5'-base (G-1) not found in other tRNAs. Deletion of G-1 results in at least a 250-fold reduction in the rate of histidine charging in vitro. To better understand the role of the G-1 nucleotide in defining the structure of tRNA(His), and to correlate structure with cognate amino acid charging, NMR and molecular dynamics (MD) studies were performed on the wild-type and a DeltaG-1 mutant Escherichia coli histidine tRNA acceptor stem microhelix. Using NMR-derived distance restraints, global structural characteristics are described and interpreted to rationalize experimental observations with respect to aminoacylation activity. The quality of the NMR-derived solution conformations of the wild-type and DeltaG-1 histidine microhelices (micro helix(His)) is assessed using a variety of MD-based computational protocols. Most of the duplex regions of the acceptor stem and the UUCG tetraloop are well defined and effectively superimposable for the wild-type and DeltaG-1 mutant microhelix(His). Differences, however, are observed at the end of the helix and in the single-stranded CCCA-3' tail. The wild-type microhelix(His) structure is more well defined than the mutant and folds into a 'stacked fold-back' conformation. In contrast, we observe fraying of the first two base pairs and looping back of the single-stranded region in the DeltaG-1 mutant resulting in a much less well defined conformation. Thus the role of the extra G-1 base of the unique G-1:C73 base pair in tRNA(His) may be to prevent end-fraying and stabilize the stacked fold-back conformation of the CCCA-3' region.

Figure 1

Sequence of E.coli tRNA^His, wild-type microhelix^His and a ΔG-1 microhelix^His variant used in this study. The unique G-1:C73 base pair is boxed.

Figure 2

NOESY spectra at 25°C, 800 MHz and 150 ms mixing time for (A) wild-type and (B) ΔG-1 mutant microhelix^His in D₂O. The positions of cross-peaks corresponding to nucleotides discussed in the text are labeled to the left of the cross-peak. The arrow indicates a weak interstrand NOE present only in the mutant.

Figure 2

NOESY spectra at 25°C, 800 MHz and 150 ms mixing time for (A) wild-type and (B) ΔG-1 mutant microhelix^His in D₂O. The positions of cross-peaks corresponding to nucleotides discussed in the text are labeled to the left of the cross-peak. The arrow indicates a weak interstrand NOE present only in the mutant.

Figure 3

Imino region of ¹H spectrum at 800 MHz in 90% H₂0/10% D₂O for (A) wild-type and (B) ΔG-1 mutant microhelix^His obtained at 25 and 5°C, respectively. The imino region of the ΔG-1 mutant at 25°C is shown in the inset to (B).

Figure 4

NMR structures of (A) wild-type and (B) ΔG-1 mutant microhelix^His. The average structures over the last 200 ps of MD simulations with full NMR restraints generated using protocol 3 are shown. Hydrogen atoms are not displayed.

Figure 5

Schematic representations of base orientations in the single-stranded region and the first two base pairs in the acceptor stem of (A) wild-type and (B) ΔG-1 mutant histidine microhelices. Riboses are represented as pentagons and bases as rectangles. Bold pentagons represent C2′-endo puckering of the sugar and C3′-endo otherwise. Bold rectangles represent bases in syn orientation about the glycosidic bond and anti otherwise. Shaded rectangles indicate bases oriented in the same plane and unshaded rectangles indicate that the base is oriented behind the plane. Base pairing is shown as horizontal dashed lines.

Figure 6

Structures of wild-type (top) and ΔG-1 mutant (bottom) histidine microhelices showing just the single-stranded region and the first two base pairs in the acceptor stem. (A) Structures generated at the end of protocol 2; (B) structures generated after completely relaxing NMR restraints at the rate of 10% every 200 ps; (C) structures generated by reimposing NMR restraints to the 20% restraint-reduced structure. Hydrogen atoms are not displayed.

Figure 7

Plots of NMR restraint violations versus time for (A) wild-type and (B) ΔG-1 mutant histidine microhelices. The structures used for calculating restraint violations were taken at 200 ps intervals from the MD simulations in which the restraint weights were reduced and then increased (protocol 3). Restraint violations are in angstroms and are shown by the lines. Restraint weights are in fractions and are indicated by the vertical bars.