Aminoacylation of 3' terminal tRNA-like fragments of turnip yellow mosaic virus RNA: the influence of 5' nonviral sequences.

The present model of the L-shaped tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA encompasses 82 nucleotides. A previous kinetic study on 3' terminal TYMV RNA fragments that contain the tRNA-like structure and a 5' nonviral GGGAGA sequence, suggested that viral sequences upstream of the tRNA-like domain, i.e., upstream of nucleotide 82, increase the rate of aminoacylation (Dreher et al. (1988) Biochimie 70, 1719-1727). Here we report an increase in the aminoacylation rate when the number of nonviral nucleotides at the 5' end of TYMV RNA transcripts was reduced. The influence of these 5' proximal nonviral sequences on the conformation of the RNA molecule was investigated by structure mapping experiments. A structure that deviates from the tRNA-like structure was found in some of the transcripts. The formation of this alternative structure is dependent upon: (1) the nature and number of the nonviral nucleotides; (2) the number and secondary structure of viral nucleotides between the nonviral nucleotides and the tRNA-like domain. Footprinting experiments with valyl-tRNA synthetase from yeast suggest that the enzyme does not recognize the alternative structure.

Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability.

Site-directed mutations were introduced in the connecting loops and one of the two stem regions of the RNA pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. The kinetic parameters of valylation for each mutated RNA were determined in a cell-free extract from wheat germ. Structure mapping was performed on most mutants with enzymic probes, like RNase T1, nuclease S1 and cobra venom ribonuclease. An insertion of four A residues in the four-membered connecting loop L1 that crosses the deep groove of the pseudoknot reduces aminoacylation efficiency. Deletions up to three nucleotides do not affect aminoacylation or RNA pseudoknot formation. Deletion of the entire loop abolishes aminoacylation. Although elimination of the pseudoknot is presumed, this could not be demonstrated. Unlike the mutations in loop L1, all mutations in the three-membered connecting loop L2 that crosses the shallow groove of the RNA pseudoknot decrease the aminoacylation efficiency considerably. Nonetheless, the RNA pseudoknot is still present in most mutated RNAs. These results indicate that a number of mutations can be introduced in both loops without abolishing aminoacylation. Results obtained with the introduction of mismatches and A.U base-pairs in stem S1 of the pseudoknot, containing three G.C base-pairs in wild-type RNA, indicate that the pseudoknot is only marginally stable. Our estimation of the gain of free energy due to the pseudoknot formation is at most 2.0 kcal/mol. The pseudoknot structure can, however, be stabilized upon binding the valyl-tRNA synthetase.

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting.

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

A functional role for the conserved protonatable hairpins in the 5' untranslated region of turnip yellow mosaic virus RNA.

The 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buitenhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). Here, we present a functional analysis of the 5' UTR of turnip yellow mosaic virus (TYMV) RNA, which contains two protonatable hairpins with nearly identical internal loops. Mutations were introduced in an infectious cDNA clone, and T7 RNA transcripts were used to infect Chinese cabbage plants. Different symptoms were observed for the various mutants, pointing to a functional role of the C-C and C-A mismatches in the hairpins of the 5' UTR. The replication of the virus is influenced by the mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influenced by the secondary structure of the leader. Various mutants were propagated for up to five serial passages of infection, and the sequence of the 5' UTR was determined. This resulted in virus RNA with new non-wild-type sequences that produced the wild-type phenotype in infected plants. Remarkably, in all cases C-C or C-A mismatches were introduced. The internal loop of the 5'-proximal hairpin seems to be more important for the viral life cycle than that of the second hairpin. A deletion of 75% of the leader, including the two hairpins, resulted in a virus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5' UTR in viral packaging is proposed.

In vitro transcription by the turnip yellow mosaic virus RNA polymerase: a comparison with the alfalfa mosaic virus and brome mosaic virus replicases.

Recently, we showed that the main determinant in the tRNA-like structure of turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitro is the 3' ACCA end. By mutational analysis of the 3'-terminal hairpin, we show here that only a non-base-paired ACCA end is functional and that the stability of the wild-type 3'-proximal hairpin is the most favorable, in that it has the lowest DeltaG value and a high transcription efficiency. With a nested set of RNA fragments, we show that the minimum template length is 9 nucleotides and that transcription is improved with increasing the length of the template. The results also suggest that proper base stacking contributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. However, the position of the internal initiation site in the template is important, and competition between the different sites takes place. Internal initiation was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AlMV). The BMV polymerase can start internally on ACCA sequences, though inefficiently. Unexpectedly, the polymerases of both AlMV and BMV can start efficiently on an internal AUGC sequence.

Analysis of the role of the pseudoknot component in the SRV-1 gag-pro ribosomal frameshift signal: loop lengths and stability of the stem regions.

The simian retrovirus-1 (SRV-1) gag-pro frameshift signal was identified in previous work, and the overall structure of the pseudoknot involved was confirmed (ten Dam E, Brierley I, Inglis S, Pleij C, 1994, Nucleic Acids Res 22:2304-2310). Here we report on the importance of specific elements within the pseudoknot. Some mutations in stem S1 that maintain base pairing have reduced frameshift efficiencies. This indicates that base pairing in itself is not sufficient. In contrast, frameshifting correlates qualitatively with the calculated stability of mutations in S2. The stems thus play different roles in the frameshift event. The nature of the base in L1 has little influence on frameshift efficiency. It is however required to bridge S2; deleting it lowers frameshifting from 23 to 9%. In L2, frameshift efficiency was not affected in a mutant that changed 10 to 12 bases. This makes it unlikely that the primary sequence of L2 plays a role in -1 frameshifting, in contrast to readthrough in Moloney murine leukemia virus (Wills N, Gesteland R, Atkins J, 1994, EMBO J 13:4137-4144). Deletions of 2 and 3 bases gave more frameshifting than the wild type, probably reflecting the increased stability of the pseudoknot due to a shorter loop L2. Deleting even more bases reduces frameshifting compared to wild-type levels. At this point, stress will build up in L2, and this will reduce overall pseudoknot stability.

Minimal template requirements for initiation of minus-strand synthesis in vitro by the RNA-dependent RNA polymerase of turnip yellow mosaic virus.

From mutational analysis of the 3'-terminal hairpin of turnip yellow mosaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TYMV RNA for initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) is the non-base-paired 3' ACC(A) end. Base pairing of this 3' end reduces the transcription efficiency drastically, and deletion of only the 3'-terminal A residue results in a fivefold drop in efficiency. The two C residues of the 3' ACCA end are required for efficient transcription, as shown by substitution mutations. However, the 5' A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the hairpin stem and loop upstream of the 3' ACCA end also do not interact with the RdRp in a base-specific way. However, for efficient transcription, the hairpin stem should be at least five bp in length, while the calculated deltaG value should be less than -10.5 kcal/mol. Unexpectedly, the use of nonstructured C-rich RNA templates showed that the RdRp can start internally on an NCCN or NUCN sequence. Therefore, a possible function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.