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UUG is used. In a representative of Gram-positive bacteria, Bacillus subtilis, 30 % of the starts are with GUG and UUG, and UUG is even more common than GUG. There are exceptional cases in bacteria where initiation takes place at AUU, AUA, ACG and CUG. These rare starting triplets are "weak" initiation codons and seemingly may play this role only in combination with "strong" Shine-Dalgarno sequences upstream and/or other initiation-promoting structural elements.

In addition to the Shine-Dalgarno sequence and the initiation codon, the ribosome-binding site (RBS) of bacterial mRNA comprises both upstream and downstream sequences and covers about 55 to 60 nucleotides, from position around -35 to position +20 (relative to the first nucleotide of the initiation codon). The sequences beyond the Shine-Dalgarno element and the initiation triplet have been reported to be non-random and also contain information that is essential for efficient initiation of translation. In particular, A and U are favored throughout all the RSB, and A is especially preferred downstream from the Shine-Dalgarno element. On the contrary, C is counter-selected in the RBS. Some optimal RBS sequence contexts can be deduced from the data available. For example, the frequent occurrences of A at position -3, GCU and AAA triplets as the codons following the initiation codon, often with A as the next nucleotide, and the sequence UUAA in the fourth and fifth codons have been mentioned.

In any case the region upstream of the Shine-Dalgarno sequence has been repeatedly reported to be important for an efficient initiation. First of all, it must be unstructured in order to facilitate some necessary contacts with the initiating ribosomal particle. Up to 20-30 nucleotides upstream from the first Shine-Dalgarno residue may be involved in these stimulatory contacts. The existence of a stable hairpins in this pre-SD region, even when the SD sequence and the initiation codon are not included, may block the initiation of translation. On the other hand, initiation "enhancers" can exist upstream of the Shine-Dalgarno element (for review, see McCarthy & Gualerzi, 1990). The well known enhancing sequence is the Epsilon motif UUUAACUUUA in highly expressed late mRNAs of bacteriophage T7 and some other phages, as well as the Epsilon-like upstream sequences in some bacterial mRNAs (e.g., UUUUAACU and UAAUUUAC in atpE cistron of atp polycistronic mRNA of E. coli, see Section 16.2).

While the Shine-Dalgarno sequence of mRNA is complementarily bound with the 3'-terminal sequence of the 16S RNA, other regions of RSB are believed to interact also with the ribosomal RNA elements of the 30S subunit (McCarthy & Brimacombe, 1994). Thus, according to chemical cross-linking experiments, the region between the Shine-Dalgarno sequence and the initiation codon is in contact with A665 in side loop of helix 22 (domain II), A1360 in end loop of helix 43 (domain III) and G1530 near the base of the ultimate helix 45 of the 16S RNA (see Fig. 9.3). It is interesting that these positions are in the regions of the secondary structure that universally conserved in all ribosomes, including eukaryotic ones. In addition to the bases of the Shine-Dalgarno sequence, the bases of mRNA 10 nucleotides and 20-21 nucleotides upstream of the SD have been reported to be protected against chemical modifications due to the interaction with the ribosome (probably with the 16S RNA).

The portion of RSB downstream of the initiation codon, however, does not display such contacts with specific positions of the 16S RNA, until the initiator tRNA is bound. Only after F-Met-RNA is bound, the downstream regions of RBS become both protected against the hydroxyl attack on the sugar-phosphate backbone and specifically cross-linkable with the 16S RNA. The cross-linkable sites of the 16S RNA seem to be all in the mRNA-binding cleft of the 30S subunit; these are C1395-C1402 in the section between domain III and the long compound hairpin (helix 44) of the 3'-terminal sequence, A532 in the end loop of helix 18 (domain I), and U1052 in helix 34 (domain III) (see Fig. 9.3). The 16S RNA nucleotides cross-linkable with the downstream region of RBS are universally conserved among ribosomes. The observation that the specific contacts of the downstream region of RBS with the mRNA-binding cleft are established only after F-Met-RNA is bound suggests that the mRNA retained by the 30S subunit prior to its fixation by codon-anticodon interaction is rather movable; probably, when it is bound only by ASD, it can slide around on the ribosome by virtue of the flexibility of the 3'-terminal region of the 16S RNA ("stand-by complex").

15.2.3.Prokaryotic Initiator tRNA

The initiation codons of prokaryotic mRNAs are recognized by formylmethionyl-tRNA (F-Met-tRNA), with its anticodon CAU (Marcker & Sanger, 1964; Clark & Marcker, 1966). Thus, the codon-anticodon pairing during initiation may be either fully complementary (AUG), in terms of the classical Watson-Crick complementarity, or partially complementary ("two-of-three"). "Wobbling" at the first position of the initiation codon (GUG and UUG) seems to be the most allowable among the partially complementary codons. (Note that this wobbling principally differs from the Crick wobbling at the third position of codons during elongation).

The structure of the initiator tRNA is organized similarly to that of the elongator tRNAs: the secondary structure can be presented as a typical clover-leaf fold (Fig. 15.5), and the hairpins are arranged into the L-shaped pattern (like in Fig. 3.7), resulting in the formation of a compact molecule with the anticodon and acceptor protuberances, akin to the paradigmatic yeast tRNAPhe (see Figs. 3.8 and 3.10) (Woo et al., 1980). Some structural differences, however, may be of principal importance for the functions of the initiator tRNA. First of all, the 5'-terminal nucleotide residue of the initiator tRNA does not form a Watson-Crick base pair with the 72nd nucleotide residue of the 3'-terminal region (Fig. 15.5). It appears to provide greater flexibility for the acceptor end; according to the crystallographic model, the 3'-end of tRNAfMet curls back towards the 5'-end and does not continue the helical organization of the acceptor stem as in the case of the elongator tRNA species. Thus the formylmethionyl residue may lie on the acceptor stem helix and contribute to the specific recognition of F-Met-tRNA by IF2.

Second, the structure of the anticodon loop in the initiator tRNAfMet displays deviations from the classical conformation in yeast tRNAPhe: the invariant U in position 33 adjacent to anticodon from the 5'-side is turned outside (whereas it turns inside the loop and faces the phosphate of the third anticodon residue in the elongator tRNA, see Fig. 3.8). For this reason the stacked conformation of the anticodon in the initiator tRNA looks distorted compared to the anticodon of the elongator tRNA. The unique presence of the stack of three consecutive G:C base pairs at the end of the anticodon helix (Fig. 15.5) in all initiator tRNAs seems to contribute to a disordered conformation of the anticodon loop.

Third, the nucleotide residues of the dihydrouridylic loop of the initiator tRNA (positions 16 and 17) are more tightly packed together and with the core of the tRNA, compared to elongator tRNA species where they appear to be loosely accommodated near the corner of the L-shaped molecule.

The differences mentioned may have concern to the special functional features of the initiator tRNA: (1) it is recognized by methionyl-tRNA transformylase (see below); (2) it is not recognized by EF-Tu in the aminoacylated form, but instead recognized by IF2 (see below); (3) it primarily settles in the P site, rather than in the A site of the ribosome.

The initiator tRNA has a specific affinity to the normal methionyl-tRNA synthetase and, correspondingly, capable of accepting methionine. Therefore, two classes of tRNAMet (Fig. 15.5) are acylated with methionine by the same aminoacyl-tRNA synthetase: the elongator tRNA recognizing the methionine codon AUG in the course of elongation, and the initiator tRNA which can recognize the AUG triplet, as well as GUG and UUG (and rarely AUU. AUA, etc.), during initiation. In contrast to Met-tRNA, the initiator Met-tRNA serves as a substrate for a special formyl transferase which transfers the formyl group from the formyl tetrahydrofolate to the amino group of the methionine residue, yielding formylmethionyl-tRNA:

The unpaired end and/or a resultant conformational feature seems to be required for the recognition of Met-tRNA by the methionyl-tRNA formylase. In its turn, the formylation of the amino group prevents the interaction with EF-Tu. EF-Tu, however, poorly interacts with non-formylated Met-tRNA either, this suggesting the presence of a structural element in tRNA moiety which also hinders the interaction with EF-Tu. The altered structure of the contact area between D- and T-loops may be such an element. The distorted structure of the anticodon loop may be relevant to the primary positioning of F-Met-tRNA at the P site and to the unusual wobble interactions of its anticodon with a variety of initiation codons (Varshney et al., 1993; Dyson et al., 1993).

In its aminoacylated and formylated state F-Met-tRNA plays a key role in initiation.

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