Gaa Ucc Gga Gug Uaa Gaa Aug

Again, the translational coupling has been shown to be tight and efficient (Schuemperli et al., 1982) this being consistent with the fact that the products of galT and galK form an enzymatic complex with equimolar proportion of the two proteins (galactose-1-phosphate uridyltransferase and galactokinase, respectively). The mutational analysis demonstrated that reinitiation at galK is reduced when termination of the preceding cistron occurs outside the initiation region (upstream of the Shine-Dalgarno sequence or downstream of the initiation codon), and the more the distance between them the less is the efficiency of reinitiation.

The translation of the message encoding for the lysis peptide (L) within the bacteriophage MS2 RNA (see below, Fig. 16.4) is also tightly coupled with the translation of the preceding coat protein cistron (C). Due to structural reasons (involvement of the RBS in relatively stable secondary structure; see Fig. 16.6, helix II), no independent initiation at the L cistron is possible. The termination at the end of the C cistron has been shown to be an absolute prerequisite for the initiation (reinitiation) at the L cistron (Adhin & van Duin, 1989). At the same time the efficiency of reinitiation in this case is low resulting in much less production of the lysis peptide as compared with that of the coat protein. This correlates with a relatively long distance between the termination codon of the C cistron and the reinitiation site of the L cistron, the latter being at more than 40 nucleotides upstream of the C cistron terminator UAA (see Fig. 16.5).

Thus, the sequential coupled translation of polycistronic mRNAs implies that the ribosomal particles (seemingly the 30S subunits) after termination are capable of "phaselessly wandering" (Sarabhai & Brenner, 1967) along mRNA around the termination site both downstream and upstream of it. Physically this phenomenon seems to be a lateral (two-dimensional) diffusion of non-translating, mRNA-associated ribosomal particles along mRNA. If they encounter a structurally available initiation site including the Shine-Dalgarno sequence and the initiation codon with a proper distance between them, they reinitiate translation. When the initiation site overlaps the termination codon of the preceding cistron the reinitiation can be very efficient and approach 100 %; this is the case of equimolar production of the encoded polypeptides. At the same time, a high probability of the dissociation of the non-translating particles from mRNA exists during the "phaseless wandering", so that the more the distance between the termination codon of the preceding cistron and the RBS of the following cistron, the less is the efficiency of the reinitiation (Adhin & van Duin, 1990). A weak RBS can be another factor reducing the efficiency of the reinitiation. Therefore, even in the case of tight coupling (strong dependence of the translation of an internal cistron on the translation of the preceding cistron), the efficiency of reinitiation after termination may vary depending on the intercistronic region: in many cases a significant portion of terminated ribosomes dissociate from mRNA, and only a part of the particles reinitiates. Correspondingly, the protein production of the downstream cistron will be decreased as compared with that of the preceding cistron.

Generally, the reinitiation by mRNA-associated ribosomal particles is more efficient than the initiation by free ribosomes. That is why relatively weak or not well exposed initiation sites can be used for reinitiation whereas the initiation by free ribosomes at them is absent or poor. Even in the case when RBS is buried in a stable secondary structure, the termination of a preceding cistron within the RBS allows the terminating ribosomes to be immediately captured by the initiation sequences (SD and AUG). This can be considered as a local rearrangement of a termination complex into an initiation complex.

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