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Figure 16.5. The nucleotide sequence of the L cistron of the bacteriophage MS2 RNA (beginning with nucleotide residue 1678) (J.F. Atkins, J.A. Steitz, C.W. Anderson & P. Model, Cell 18,247-256,1979; M.N. Beremand & T. Blumenthal, Cell 18, 257-266,1979). The amino acid sequence of the lysis protein is written above the nucleotide sequence, whereas the amino acid sequences of the end of the coat protein and the start of the replicase subunit polypeptide are given below the nucleotide sequence.

protein synthesis. After the translation of the C cistron has begun, ribosomes move along its coding sequence in the direction of the S cistron and unfold the secondary and tertiary structure in the course of their progression. This results in the opening of the S cistron initiation region (Fig. 16.6) (see also Section 16.3.1). Hence, even before the first ribosome has completed translation of the C cistron and, thus, before the first coat protein molecule has been synthesised, the initiation region of the S cistron becomes accessible for initiation and, correspondingly, synthesis of the RNA replicase subunit is initiated.

In order to form the active RNA replicase molecule, the product of the S cistron translation must associate with three host cell proteins. Two of these proteins are the elongation factors EF-Tu and EF-Ts, and the third is the ribosomal protein S1. In other words, the complete active RNA replicase is a protein with a quaternary structure consisting of four different subunits (R, S1, EF-Tu and EF-Ts), and only one of them (R) is coded by the phage RNA. The RNA replicase is a template-specific RNA-dependent RNA polymerase using the original chain ("+" chain) of the MS2 RNA to form the complementary chain ("-" chain) and then, using it as a template, to produce numerous copies of the original "+" chain.

The RNA replicase has been also found to be a repressory protein in translation of MS2 RNA: it specifically recognises the RBS of the C cistron, binds to it and blocks the initiation (Weber et al., 1972; Meyers et al., 1981). This happens during early stage of infection when the concentration of the replicase increases and eventually reaches a certain level. The repression of the C cistron translation at this stage is aimed to avoid the situation where the RNA serves at same time as template for translation by ribosomes and for replication by the enzyme, i.e., to "clear" the RNA of ribosomes. As the replication proceeds, new RNA molecules appear and exceed the number of the RNA replicase molecules, so that the coat protein molecules start to be produced again from non-repressed C cistrons.

The completion of the C cistron translation by the ribosomes results in the appearance of free coat protein molecules. As translation proceeds, and as new MS2 RNA molecules becomes available for the translation, this protein accumulates; eventually it will be used in the self-assembly of mature page particles. The coat protein, however, in addition to its role in phage particle assembly, possesses a strong specific affinity to the region of MS2 RNA between the C and S cistrons, including the RBS of S cistron (see Fig. 16.4). The protein binds to this region (operator) and represses the initiation of the S cistron translation (Lodish & Zinder, 1966; Bernardi & Spahr, 1972). The repression seems to result from the labile secondary structure (Fig. 16.6 helix IV) being stabilised by the phage coat protein, and thus the Shine-Dalgarno sequence and the initiation codon of the S cistron becoming inaccessible for ribosomes. Hence, after the translation of the S cistron has been allowed by the translation of the preceding cistron, the S cistron translation is repressed due to accumulation of the protein coded by the preceding cistron. The repression of the further synthesis of this protein prevents an unnecessary overproduction of the enzyme. In this way the phage coat protein, which plays the part of the S cistron repressor, performs the regulatory function in translation.

The A cistron cannot be translated until MS2 replication is started. Its initiation region is masked by the structure of the intact MS2 RNA. It may be exposed for in vitro translation and thus can be initiated as a result of some artificial treatments, e.g., partial nuclease or heat-induced degradation of the intact polynucleotide chain, or mild treatment with formaldehyde, which disrupt base pairings. In the course of RNA replication, however, at the early period of "+" chain formation when the chain is still growing, the three-dimensional structure of the 5'-terminal section containing the RBS of the A cistron has not yet been fully formed. It is this period that seems to be used for initiating translation of the A cistron under normal conditions (Robertson & Lodish, 1970; Kolakofsky & Weissmann, 1971). Since the mature virus particle contains only one molecule of the A protein per 180 molecules of the coat protein, the relatively brief period during which the initiation of the A cistron translation is possible appears to be sufficient for the required production of the A protein. Thereafter, the elongated MS2 RNA folds in such a way that the initiation region of the A cistron becomes involved in some three-dimensional structure which makes the RBS inaccessible for free ribosomes.

Thus, the translation of MS2 RNA provides examples of several different regulatory systems. First of all, the interaction of the RNA replicase molecule with the initiation site of the C cistron and the binding of the coat protein molecules to the initiation site of the S cistron represent a typical translational repression mechanism. In addition, translational coupling via opening RBS by translation of a preceding cistron (Section 16.3.1), translational coupling via reinitiation (Section 16.3.2) and coupled replication-translation are observed during expression of the polycistronic MS2 RNA.

16.4.2.Regulation of Translation of Ribosomal Protein mRNAs

The bacterial cell is known to avoid overproduction of the ribosomal proteins. Generally speaking, the ribosomal proteins are synthesised in amounts required just for ribosomal assembly, in accordance with the amount of ribosomal RNA formed; under normal conditions the cell contains no significant excess of free ribosomal proteins The co-ordinated levels of production of nearly all ribosomal proteins in equimolar amounts are achieved even though their genes are not organised as a single regulated block, but are represented by approximately 16 independent operons, which are scattered throughout the cell genome. The co-ordinated and virtually stoichiometric production of the ribosomal proteins and the prevention of their overproduction are maintained by a controlling mechanism which provides the repression of translation by protein excess (translational feedback control) (Dean & Nomura, 1980).

A large proportion of the genes coding for ribosomal proteins (31 out of 52) are present in two main clusters on the E. coli chromosome. One of these clusters is located in the str-spc region at the 72nd min, and the other in the rif region at the 89th min. The str-spc region contains four operons coding for 27 ribosomal proteins, EF-Tu, and EF-G, as well as for the a-subunit of RNA polymerase. The rif region contains two operons coding for four ribosomal proteins, as well as for the ß- and ß'-subunits of RNA polymerase. Each operon produces a polycistronic mRNA. The cistrons and their order in these polycistronic mRNAs are shown schematically in Fig. 16.7.

Studies conducted by several groups, first of all by Nomura and ssociates, in 1979-1982 (for reviews, see Nomura et al., 1982, 1984; Lindahl & Zengel, 1982, 1986; Draper, 1987) demonstrated that in the case of each polycistronic mRNA, one of the translation products, a ribosomal protein, serves as a repressor of the translation of a corresponding mRNA (these products are circled in Fig. 16.7). This effect has been demonstrated both in experiments in vivo and in cell-free systems. Experiments in vivo have shown that the synthesis of the ribosomal proteins coded by the corresponding mRNA is inhibited when the overproduction of one of the proteins circled in Fig. 16.7 is induced. Induction of proteins S7, L4, S8, S4, L1, or L10 results in the inhibited synthesis of only those ribosomal proteins that are coded by the polycistronic mRNA possessing the cistron of the corresponding protein. Experiments in vitro have brought even more direct results: adding one of the above proteins, e.g. S7, L4, S8, S4, L1, or L10, to the cell-free translation system leads to a selective inhibition of synthesis of only that set of proteins which is coded by the mRNA containing the cistron corresponding to the added protein.

Synthesis of some of the proteins coded by the listed polycistronic mRNAs, however, is not inhibited when the repressory ribosomal proteins are added. Protein S12, for example, continues to be synthesised after the protein S7 is added to the cell-free system or in the in vivo version of the experiment with the selective induction of protein S7. Similarly, the synthesis of proteins L14 and L24 does not stop in response to the addition or overproduction of protein S8. It is noteworthy that the cistrons of the proteins not controlled by proteins S7 or S8 are found to be proximal to the 5'-end of the polycistronic mRNA.

All of these observations may be best explained by assuming that the repressor protein binds specifically to the initiation region of one cistron and blocks the translation of all cistrons located in the direction of the 3'-end. Protein S8, for example, binds with the origin of the cistron of protein L5 and, as a result, the translation of all subsequent downstream-located (but not upstream-located) cistrons is repressed. The implication is that in these cases free ribosomes cannot initiate the translation of each cistron independently. The sequential translation via reinitiation appears to occur instead: ribosomes that have terminated the translation of the preceding cistron do not dissociate from the template but pass directly to reinitiation at the next cistron. Such sequential translation of cistrons provides for the equimolar production of ribosomal proteins coded by a given polycistronic mRNA.

There are some exceptions, however, in the same polycistronic mRNAs. For example, it has been shown that the translation of mRNA cistrons for EF-Tu and for RNA polymerase subunits ß + ß' and a is not repressed by proteins S7, L10, and S4, respectively (Fig. 16.7). Therefore, it appears that the initiation regions of these mRNA cistrons are capable of binding the free ribosomes, providing for independent initiation of translation. On the other hand, it is known that in the case of the synthesis of proteins L10 and L7/L12, the production of protein L7/L12 is four times greater than the production of protein L10, in accordance with their stoichiometry in the ribosome; the independent initiation of translation of the L7/ L12 cistron takes place here and the initiation rate for this cistron is far greater than that for the L10 cistron. At the same time, as has already been indicated, protein L10 represses the translation of both proteins L10 and L7/L12: the repression of its own cistron prevents the opening of the RBS of the next L7/

L12 cistron (see Section 16.3.1).

Identifying the attachment sites (operators) of the repressory ribosomal proteins on polycistronic mRNAs is particularly interesting. It has been demonstrated that if the origin of the structural gene for protein S13 and the preceding nucleotide sequence is deleted, protein S4 is unable to repress translation of the corresponding polycistronic mRNA (fourth line in Fig. 16.7) (Nomura et al., 1980). In contrast, protein S7 can repress its own synthesis if the leader of its polycistronic mRNA (first line in Fig. 16.7), including the S12 cistron, is absent (Dean et al., 1981). Protein L1 has been shown to exert repressory action upon its bicistronic mRNA (fifth line in Fig. 16.7) only in the presence of the 5'-terminal sequence, preceding the

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