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Figure 15-3 RNA secondary structure in the central part of MS2 RNA. Start codons of coat, lysis, and replicase are boxed. Numbers in dashed hairpins indicate how many nucleotides are not drawn. Structures shown are instrumental in the translational coupling between coat gene on the one hand and lysis and replicase genes on the other. MJ and VD are long-distance interactions.

secondary structures. To understand this it is useful to consider the forces that drive the binding of ribosomes to translational start regions on prokaryotic messenger RNAs. There are at least three contributors to this binding energy: (i) base complementarity between 16S ribosomal RNA and the Shine-Dalgarno sequence just upstream of the start codon on the messenger RNA; (ii) interaction of the antico-don on the initiator fmet-tRNA with the AUG start codon on the messenger RNA; and (iii) binding of ribosomal protein S1 to pyrimidine-rich sequences frequently found upstream of the Shine-Dalgarno sequence (81). If a strong pre-existing secondary structure in messenger RNA prevents binding of one or more of the above three components, there will be no ribosome binding and therefore no initiation of protein synthesis (24).

Experiments have shown that if the start codon of the coat gene is deleted or mutated, preventing ribosomes from translating this gene, neither lysis nor replicase protein is synthesized (9, 25, 69). This is because the beginning of the lysis and replicase genes lie within RNA secondary structures that are too stable to allow ribosome binding (figure 15-3).

Control of Replicase Gene Translation

Given that a ribosome contacts messenger RNA over a stretch of about nucleotides 20 upstream and 15 nt downstream of the initiator AUG, it is clear that three regions of secondary structure around the replicase start site (figure 15-3) could contribute to impeding the ribosome from binding to that site. These are first the long-distance interaction, MJ, second, the operator hairpin containing the AUG itself, and finally stem R32. When base-pairing at stem MJ is abolished by the introduction of mismatches or by deleting the sequence 1427-1434, there is a large increase in replicase synthesis, which is independent of translation of the coat gene (9, 93). Apparently, the remaining operator and R32 hairpin structures are together not strong enough to block the entry of ribosomes. The coupling then works like this. Every time a ribosome reads the coat gene it disrupts base-pairing at stem MJ. Once this happens, other ribosomes can bind to the replicase start site and initiate translation there, even though this site is some 340 nt downstream from the position where the ribosome is translating the coat protein gene. Further support for this model comes from the finding that introduction of translational stop codons into the coat protein gene upstream of nt 1427 inactivates translation of the replicase gene (the ribosome never gets to stem MJ). However, stop codons placed beyond nt 1434 allow replicase synthesis to proceed as efficiently as it does in wild-type RNA. Furthermore, the more frequently the coat-protein gene is translated, the more replicase is produced (S. H. E. van den Worm and J. van Duin, unpublished results). Coat-replicase coupling also exists in Qb RNA, but has not been studied in great detail.

There is a second level of control of translation of the replicase gene. When the concentration of coat protein becomes sufficiently high in the cell, dimers bind to the operator hairpin, precluding further translational starts of the replicase gene by excluding ribosome binding. This protein-RNA interaction is maintained in the intact virion and has been studied exhaustively (29, 89, 90, 102, 103). It is also believed to stimulate capsid formation by serving as a nucleation site for further addition of coat dimers.

Control of Lysis Gene Translation

Independent access of ribosomes to the lysis gene start site (nt 1678) is precluded by the "lysis hairpin'' (figure 15-3). In the absence of coat gene translation, the lysis gene is not expressed unless the lysis hairpin has been destabilized by disruption of base pairs (10). Surprisingly, activation of the lysis gene is not directly coupled to opening of this hairpin by ribosome movement over the overlapping coat gene (as with the replicase gene). Instead, translation starting at the lysis gene depends on termination of translation at the end of the coat gene (nt 1725). If this UAA stop codon (and the UAG stop codon that immediately follows it) are mutated so that termination does not take place until nucleotide 1749 (UGA), there is no expression of the lysis gene. On the other hand, if stop codons in the coat gene are introduced by mutagenesis between nt 1678 and 1725 or even not too far upstream of the lysis start, lysis protein is made. In fact, the closer the coat gene stop codon is placed to the lysis start codon, the more lysis protein is made (10). These results suggest that ribosomes that have reached the coat stop codon and finished translation can reinitiate at the lysis start codon after randomly drifting a short distance from their site of termination. From the ratio in which coat and lysis proteins are synthesized by phage MS2, it follows that the probability to back up to the lysis start and successfully reinitiate is only 5% in the wild-type situation.

This "scanning" model was further tested by introducing an additional start codon a short distance downstream from the authentic lysis start. Now, ribosomes used the new start codon in place of the authentic one, as it was closest to the termination triplet and thus formed a barrier that prevented ribosomes from reaching the authentic start site. If, however, coat gene termination was engineered upstream of the lysis start codon, the authentic initiation site was again used, because now this one was again closest to the termination site (1). Independent support for the scanning model comes from in vitro experiments with short messenger RNAs containing only 5 codons. Here, the ribosome shuffles back and forth between the start and stop codons, making multiple copies of a pentapeptide without ever leaving the template (65). Reinitiation is a commonly used mechanism to translate the distal reading frames in polycis-tronic messenger RNA. It is not known what determines the efficiency of these restarts. Ribosomes are released from the messenger RNA if no reinitiation sites are nearby.

Control of Maturation Protein Synthesis

In figure 15-4B the equilibrium secondary structure of the 5' untranslated region (UTR) of MS2 RNA is shown. A strong stem-loop at the 5' end is followed by a "cloverleaf structure'': three hairpins enclosed by a long-range interaction, formed here by the Shine-Dalgarno (SD) sequence of the maturation gene, base-paired to an upstream complementary sequence (UCS). This long-distance interaction effectively prevents ribosome binding (30). How then is the maturation protein made? The answer is that ribosomes can only bind to the maturation start site on RNA chains that are in the process of being synthesized and have not yet reached the equilibrium folding.

Consider a growing (+) strand RNA being made on a (—) strand RNA template. By the time nucleotide 123 has emerged from the polymerase complex, all nucleotides needed to build the cloverleaf shown in figure 15-4B are present. However, translation cannot yet start because stable ribosome binding needs the start codon and sequences downstream of it, up to about nucleotide 140-145. It is known that small RNAs such as tRNA (~75 nt) fold up correctly within milliseconds (from the denatured state). Phage RNA polymerases are slow and incorporate approximately 35 nucleotides per second into a growing chain. Thus, one expects the cloverleaf structure to be formed long before the ribosome binding site is made, as addition of a further 20 nucleotides beyond nt 123 would take at least about 500 ms. However, we found that the folding of the 5' untranslated region of MS2 RNA to its final equilibrium is unusually slow: it takes minutes to reform upon its denaturation (67). Generally speaking, such a delay can u c

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