C A

tRNATrP C : G Opal codon tRNAGln U : A Amber codon

10.4.3.Principal Types of Mispairing

Analysis of miscoding in the cases of poly(U) and other template polynucleotides in vitro, as well as in vivo, including miscoding induced by various aminoglycoside antibiotics, has demonstrated that errors are largely due to G:U or U:G pairing, as well as to U:U pairing (or juxtaposition) at any position of the anticodon-codon duplex. Mispairing or juxtaposition of the U:C or C:U types are less common. Some rare errors are due to the juxtaposition of the C in the anticodon and the A of the codon. In exceptional cases errors involve the formation of A:G or G:A pairs or juxtapositions, as well as of C:C, A:A, and G:G juxtapositions.

It appears that all types of juxtapositions are possible in the wobble position, including I:G, G:A, G:G, U:U, U:C, C:A, C:U, and C:C. This probably explains why all of the isoacceptor tRNAs can recognize in the cell-free system all of the four codons of a given codon family, i.e. of codons that have the same initial two nucleotides.

10.4.4.Factors Contributing to Miscoding

In addition to aminoglycoside antibiotics, a number of less specific factors including ionic conditions of the medium can increase the number of errors in the codon-dependent entry of the aminoacyl-tRNA into the translating ribosomes, Generally, all factors increasing the affinity of the tRNA to the ribosome result in increased miscoding. Increased Mg2+ concentration in the medium and the addition of diamines (e.g. putrescine) or polyamines (e.g. spermidine) increase the level of errors during cell-free translation. Ethyl alcohol and other hydrophobic agents added in even low concentrations also increase miscoding. Urea, in contrast, leads to a decrease in the miscoding level. As for general environmental factors, a lower temperature, decreased pH, and low ionic strength also contribute to a higher miscoding.

Structural features of the tRNA itself also play a part in the accuracy of the codon-dependent binding of tRNA in the A site (for a review, see Kurland & Ehrenberg, 1984). In particular, the structure of the D hairpin may be important. For example, the mutational alteration of G to A in the D arm of tRNATrp stimulates the pairing of this tRNA (which has a CCA anticodon) with the noncognate codons UGA and UGU. There is a reason to believe that the alteration of the D arm increases the affinity of tRNA to the A site.

Furthermore, the ribosome structure plays an important role in the accuracy of aminoacyl-tRNA selection. Gorini (1971, 1974) was the first to demonstrate that certain mutations leading to alterations in ribosomal components may either decrease or increase the level of miscoding. It has been found that the mutational alterations in protein S12 resulting in resistance to streptomycin (strA mutations) confer greater fidelity to the bacterial ribosome in the codon-dependent selection of aminoacyl-tRNA. In contrast, the so-called ram-mutations (ribosome ambiguity mutations) involving protein S4 make the ribosome less selective and increase the level of miscoding. Specific mutational alterations in protein S5 also decrease the selectivity of the ribosome. Mutations in eukaryotic (yeast) analogs of these proteins have been shown to exert the same effects on the fidelity of the eukaryotic ribosome. More recently it has been demonstrated that mutational alterations of the 16S RNA of bacteria or 18S RNA of Eukaryotes (yeasts) in the regions

Figure 10.12. Kirromycin.

around the A site, namely positions 517, 912, 1054, 1409, 1495 and other (see Fig. 6.1; cited in Noller, 1991; Green & Noller, 1997) or their equivalents, also affect translational accuracy of the ribosome. It may well be that the 30S or 40S subunit components forming the A site and located nearby - particularly the above-mentioned RNA regions and the tightly-clustered group of proteins S4, S5, and S12 - are vital not only to the strength of tRNA retention but also may define the degree of structural rigidity/flexibility of the tRNA anticodon or mRNA codon positioned on the ribosome.

The environmental factors listed above, as well as intrinsic structural factors, affect the extent of the selectivity of the mRNA-programmed ribosome with respect to tRNA. At the same time, the miscoding level in the system depends not only on selectivity as defined by the intrinsic properties of the components under given conditions, but on the ratio of the components as well. It is apparent that as the ratio of the concentration of noncognate tRNAs to that of cognate tRNA increases, the probability of misbinding becomes higher. It is for this reason that, for example, when poly(U) is translated in the cell-free system and the cognate phenylalanyl-tRNA is depleted as a result of the synthesis, the incorporation of leucine, isoleucine, and other incorrect amino acids into the polypeptide tends to increase. In an extreme case the system with poly(U)-programmed ribosomes may be supplied only with leucyl-tRNA, and then pure polyleucine will be synthesized on poly(U) as a template (although, of course, at a markedly slower rate than with polyphenylalanine synthesis). In natural systems the amount of amino acids misincorporated into the synthesized polypeptide may depend greatly on the concentrations of different aminoacyl-tRNA species. Thus, cell starvation for an amino acid results in amino acids with near-coding specifities extensively replacing this amino acid in the polypeptide chains (Parker et al., 1978; Gallant & Folley, 1979). Strong effects on mistranslation were reported in the cases of overproduction of foreign proteins in bacterial cells, due to aberrations of a normal balance between different amino acids in the intracellular medium (Bogosian et al., 1990).

10.4.5.Miscoding Level In Vivo under Normal Conditions

Since miscoding depends largely on a number of environmental and structural factors, it is clear that its level in the cell-free systems varies greatly. Therefore, it is important to estimate the natural miscoding level in normal living cells that are not subjected to extreme conditions and do not carry mutations affecting the protein-synthesizing machinery.

Several attempts have been made at estimating the level of miscoding in vivo. Loftfield's classical estimates (1972; see also Coons et al., 1979) provided data on the rate of misincorporation of valine instead of threonine in the a-chains of rabbit globin; a value of about 2-6x10-4 was obtained. The frequency of cysteine misincorporation, probably instead of arginine, into the completed (folded and assembled) E. coli flagellin which normally does not contain cysteine was of the same order of magnitude - 10-4 per codon, according to the estimate of Edelman & Gallant (1977). Later more direct estimates of the miscoding level in vivo principally confirmed the values from 10-4 to 10-3 for the average frequency of translational errors per codon (for review, see Kurland et al., 1990).

Some codons, however, can be misread more frequently than the above estimated rate. In particular, miscoding within one codon group (i.e. among codons differing only in the third nucleotide) is more probable than other replacements. It follows from the code dictionary (Fig. 2.2) that the most probable amino acid replacements, resulting from miscoding, would be the following: Phe « Leu, Cys « Trp, His « Gln, Ile « Met, Asn « Lys, Ser « Arg, and Asp « Glu. It is expected that the replacements from left to right (mispairing with U or C in the third codon position) are more probable than replacements from

tRNA 1

tRNA 1

right to left (mispairing with A or G in the third codon position). Indeed, lysine substitutions for asparagine are far more frequent than replacements of lysine; similarly, histidine is often replaced by glutamine, whereas glutamine is rarely replaced. For instance, in the experiments on the in vivo translation of MS2 coat protein mRNA the frequency of misreading of the asparagine codon AAU leading to the substitution of the positively charged lysine for the uncharged asparagine was about 5x10-3 (though the frequency of misreading of the asparagine codon AAC was nearly an order of magnitude less, being about 2x10-4) (Parker et al., 1983). In exceptional cases the level for some amino acid replacements was reported to reach 10-2 per codon.

The level of miscoding is usually much higher in cell-free systems; the replacement frequency may be as great as 10-2 and, within the same codon group (Phe ® Leu), even 10-1 per codon. However, under controlled ionic conditions (in a low Mg2+ concentration and with optimal proportions of other components), a level of miscoding approaching the values of 10-4 to 10-3 errors per codon may be attained.

It is important to note that under certain conditions the level of miscoding in vivo can be greatly increased, both in bacterial and in animal cells. This can be achieved by starving for certain amino acids, as well as by adding ethyl alcohol and some other agents to the medium. As already mentioned miscoding in the cell increases in response to aminoglycoside antibiotics.

A certain miscoding level may be of great biological significance and therefore is maintained in evolution. Bacterial mutants with a low miscoding level (streptomycin-resistant mutants) can be obtained, but this level is always higher in wild strains that have been isolated from nature and are more adapted to survival. There is no doubt that, in certain circumstances, miscoding contributes to survival, e.g. in cases of mutations that would otherwise be lethal. Thus, misbinding of an aminoacyl-tRNA to the termination codon produced by the mutation of a sense codon may ensure that a functional protein molecule is completed. The result of this "deception for the sake of salvation" is that the nonsense mutant survives. Similarly, certain mutants with the point amino acid replacements in important proteins, otherwise lethal, may survive through miscoding. In addition, the cell may permanently employ an infrequent misbinding of aminoacyl-tRNA to the normal termination codon; this results in an mRNA read-through beyond its usual coding region (see Section 14.1) and therefore in a synthesis of small amounts of longer polypeptides yielding functionally differing proteins, which may be necessary to some processes in the cell. It is also possible that a cell occasionally replaces an amino acid in a similar manner in order to synthesize some needed variant of a given protein in small amounts. In any event, too great coding accuracy during translation would probably restrict the cellular flexibility necessary for survival.

Another consideration concerns the rate of protein synthesis. The point is that the accuracy requires time and energy. Therefore, the attainment of an exceedingly high accuracy will decelarate translation and exhaust energy resources of the cell (Kurland tRNA

tRNA

tRNA

tRNAjVal

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