Novel amino acids can be inserted at certain stop codons

KEY CONCEPTS:

• Changes in the reading of specific codons can occur in individual genes.
• The insertion of seleno-Cys-tRNA at certain UGA codons requires several proteins to modify the Cys-tRNA and insert it into the ribosome.
• Pyrrolysine can be inserted at certain UAG codons.

Specific changes in reading the code occur in individual genes. The specificity of such changes implies that the reading of the particular codon must be influenced by the surrounding bases.

A striking example is the incorporation of the modified amino acid seleno-cysteine at certain UGA codons within the genes that code for selenoproteins in both prokaryotes and eukaryotes. Usually these proteins catalyze oxidation-reduction reactions, and contain a single seleno-cysteine residue, which forms part of the active site. The most is known about the use of the UGA codons in three E. coli genes coding for formate dehydrogenase isozymes. The internal UGA codon is read by a seleno-Cys-tRNA. This unusual reaction is determined by the local secondary structure of mRNA, in particular by the presence of a hairpin loop downstream of the UGA.

Mutations in 4 sel genes create a deficiency in selenoprotein synthesis. selC codes for tRNA (with the anticodon ACU) that is charged with serine. selA and selD are required to modify the serine to seleno-cysteine. SelB is an alternative elongation factor. It is a guanine nucleotide-binding protein that acts as a specific translation factor for entry of seleno-Cys-tRNA into the A site; it thus provides (for this single tRNA) a replacement for factor EF-Tu. The sequence of SelB is related to both EF-Tu and IF-2 (for review see Bock, 1991).

Why is seleno-Cys-tRNA inserted only at certain UGA codons? These codons are followed by a stem-loop structure in the mRNA. Figure 7.12 shows that the stem of this structure is recognized by an additional domain in SelB (one that is not present in EF-Tu or IF-2). A similar mechanism interprets some UGA codons in mammalian cells, except that two proteins are required to identify the appropriate UGA codons. One protein (SBP2) binds a stem-loop structure far downstream from the UGA codon, while the counterpart of SelB (called SECIS) binds to SBP2 and simultaneously binds the tRNA to the UGA codon (Fagegaltier et al., 2000).

Another example of the insertion of a special amino acid is the placement of pyrrolysine at a UAG codon. This happens in both an archaea and a bacterium (Srinivasan, James, and Krzycki, 2002; Hao et al., 2002). The mechanism is probably similar to the insertion of seleno-cysteine. An unusual tRNA is charged with lysine, which is presumably then modified. The tRNA has a CUA anticodon, which responds to UAG. There must be other components of the system that restricts its response to the appropriate UAG codons.

There are sporadic alterations of the universal code

KEY CONCEPTS:

• Changes in the universal genetic code have occurred in some species.
• They are more common in mitochondrial genomes, where a phylogenetic tree can be constructed for the changes.
• In nuclear genomes, they are sporadic and usually affect only termination codons. 

The universality of the genetic code is striking, but some exceptions exist. They tend to affect the codons involved in initiation or termination and result from the production (or absence) of tRNAs representing certain codons. The changes found in principal (bacterial or nuclear) genomes are summarized in Figure 7.10.

Almost all of the changes that allow a codon to represent an amino acid affect termination codons:

• In the prokaryote Mycoplasma capricolum, UGA is not used for termination, but instead codes for tryptophan. In fact, it is the predominant Trp codon, and UGG is used only rarely. Two Trp-tRNA species exist, with the anticodons UCA (reads UGA and UGG) and CCA (reads only UGG).
• Some ciliates (unicellular protozoa) read UAA and UAG as glutamine instead of termination signals. Tetrahymena thermophila, one of the ciliates, contains three tRNA^Glu species. One recognizes the usual codons CAA and CAG for glutamine, one recognizes both UAA and UAG (in accordance with the wobble hypothesis), and the last recognizes only UAG. We assume that a further change is that the release factor eRF has a restricted specificity, compared with that of other eukaryotes.
• In another ciliate (Euplotes octacarinatus), UGA codes for cysteine. Only UAA is used as a termination codon, and UAG is not found. The change in meaning of UGA might be accomplished by a modification in the anticodon of tRNA^Cys to allow it to read UGA with the usual codons UGU and UGC.
• The only substitution in coding for amino acids occurs in a yeast (Candida), where CUG means serine instead of leucine (and UAG is used as a sense codon).

Acquisition of a coding function by a termination codon requires two types of change: a tRNA must be mutated so as to recognize the codon; and the class 1 release factor must be mutated so that it does not terminate at this codon.

The other common type of change is loss of the tRNA that responds to a codon, so that the codon no longer specifies any amino acid. What happens at such a codon will depend on whether the termination factor evolves to recognize it.

All of these changes are sporadic, which is to say that they appear to have occurred independently in specific lines of evolution. They may be concentrated on termination codons, because these changes do not involve substitution of one amino acid for another. Once the genetic code was established, early in evolution, any general change in the meaning of a codon would cause a substitution in all the proteins that contain that amino acid. It seems likely that the change would be deleterious in at least some of these proteins, with the result that it would be strongly selected against. The divergent uses of the termination codons could represent their "capture" for normal coding purposes. If some termination codons were used only rarely, they could be recruited to coding purposes by changes that allowed tRNAs to recognize them.

Exceptions to the universal genetic code also occur in the mitochondria from several species. Figure 7.11 constructs a phylogeny for the changes. It suggests that there was a universal code that was changed at various points in mitochondrial evolution. The earliest change was the employment of UGA to code for tryptophan, which is common to all (non-plant) mitochondria (for review see Osawa et al., 1992).

Some of these changes make the code simpler, by replacing two codons that had different meanings with a pair that has a single meaning. Pairs treated like this include UGG and UGA (both Trp instead of one Trp and one termination) and AUG and AUA (both Met instead of one Met and the other Ile).

Why have changes been able to evolve in the mitochondrial code? Because the mitochondrion synthesizes only a small number of proteins (~10), the problem of disruption by changes in meaning is much less severe. Probably the codons that are altered were not used extensively in locations where amino acid substitutions would have been deleterious. The variety of changes found in mitochondria of different species suggests that they have evolved separately, and not by common descent from an ancestral mitochondrial code.

According to the wobble hypothesis, a minimum of 31 tRNAs (excluding the initiator) are required to recognize all 61 codons (at least 2 tRNAs are required for each codon family and 1 tRNA is needed per codon pair or single codon). But an unusual situation exists in (at least) mammalian mitochondria in which there are only 22 different tRNAs. How does this limited set of tRNAs accommodate all the codons?

The critical feature lies in a simplification of codon-anticodon pairing, in which one tRNA recognizes all four members of a codon family. This reduces to 23 the minimum number of tRNAs required to respond to all usual codons. The use of AG^A_G for termination reduces the requirement by one further tRNA, to 22.

In all eight codon families, the sequence of the tRNA contains an unmodified U at the first position of the anticodon. The remaining codons are grouped into pairs in which all the codons ending in pyrimidines are read by G in the anticodon, and all the codons ending in purines are read by a modified U in the anticodon, as predicted by the wobble hypothesis. The complication of the single UGG codon is avoided by the change in the code to read UGA with UGG as tryptophan; and in mammals, AUA ceases to represent isoleucine and instead is read with AUG as methionine. This allows all the nonfamily codons to be read as 14 pairs.

The 22 identified tRNA genes therefore code for 14 tRNAs representing pairs, and 8 tRNAs representing families. This leaves the two usual termination codons UAG and UAA unrecognized by tRNA, together with the codon pair AG^A_G. Similar rules are followed in the mitochondria of fungi (for review see Fox, 1987).

Modified bases affect anticodon-codon pairing

KEY CONCEPTS:

• Modifications in the anticodon affect the pattern of wobble pairing and therefore are important in determining tRNA specificity.

The most direct effect of modification is seen in the anticodon, where change of sequence influences the ability to pair with the codon, thus determining the meaning of the tRNA. Modifications elsewhere in the vicinity of the anticodon also influence its pairing.

When bases in the anticodon are modified, further pairing patterns become possible in addition to those predicted by the regular and wobble pairing involving A, C, U, and G. Figure 7.8 shows the use of inosine (I), which is often present at the first position of the anticodon. Inosine can pair with any one of three bases, U, C, and A.

This ability is especially important in the isoleucine codons, where AUA codes for isoleucine, while AUG codes for methionine. Because with the usual bases it is not possible to recognize A alone in the third position, any tRNA with U starting its anticodon would have to recognize AUG as well as AUA. So AUA must be read together with AUU and AUC, a problem that is solved by the existence of tRNA with I in the anticodon.

Actually, some of the predicted regular combinations do not occur, because some bases are always modified. There seems to be an absolute ban on the employment of A; usually it is converted to I. And U at the first position of the anticodon is usually converted to a modified form that has altered pairing properties.

Some modifications create preferential readings of some codons with respect to others. Anticodons with uridine-5-oxyacetic acid and 5-methoxyuridine in the first position recognize A and G efficiently as third bases of the codon, but recognize U less efficiently. Another case in which multiple pairings can occur, but with some preferred to others, is provided by the series of queuosine and its derivatives. These modified G bases continue to recognize both C and U, but pair with U more readily.

A restriction not allowed by the usual rules can be achieved by the employment of 2-thiouridine in the anticodon. Figure 7.9 shows that its modification allows the base to continue to pair with A, but prevents it from indulging in wobble pairing with G (for review see Bjork, 1987).

These and other pairing relationships make the general point that there are multiple ways to construct a set of tRNAs able to recognize all the 61 codons representing amino acids. No particular pattern predominates in any given organism, although the absence of a certain pathway for modification can prevent the use of some recognition patterns. So a particular codon family is read by tRNAs with different anticodons in different organisms.

Often the tRNAs will have overlapping responses, so that a particular codon is read by more than one tRNA. In such cases there may be differences in the efficiencies of the alternative recognition reactions. (As a general rule, codons that are commonly used tend to be more efficiently read.) And in addition to the construction of a set of tRNAs able to recognize all the codons, there may be multiple tRNAs that respond to the same codons.

The predictions of wobble pairing accord very well with the observed abilities of almost all tRNAs. But there are exceptions in which the codons recognized by a tRNA differ from those predicted by the wobble rules. Such effects probably result from the influence of neighboring bases and/or the conformation of the anticodon loop in the overall tertiary structure of the tRNA. Indeed, the importance of the structure of the anticodon loop is inherent in the idea of the wobble hypothesis itself. Further support for the influence of the surrounding structure is provided by the isolation of occasional mutants in which a change in a base in some other region of the molecule alters the ability of the anticodon to recognize codons.

Another unexpected pairing reaction is presented by the ability of the bacterial initiator, fMet-tRNA_f, to recognize both AUG and GUG. This misbehavior involves the third base of the anticodon.

tRNA contains modified bases

KEY TERMS:

• Modification of DNA or RNA includes all changes made to the nucleotides after their initial incorporation into the polynucleotide chain.

KEY CONCEPTS:

• tRNAs contain >50 modified bases.
• Modification usually involves direct alteration of the primary bases in tRNA, but there are some exceptions in which a base is removed and replaced by another base. 

Transfer RNA is unique among nucleic acids in its content of "unusual" bases. An unusual base is any purine or pyrimidine ring except the usual A, G, C, and U from which all RNAs are synthesized. All other bases are produced by modification of one of the four bases after it has been incorporated into the polyribonucleotide chain.

All classes of RNA display some degree of modification, but in all cases except tRNA this is confined to rather simple events, such as the addition of methyl groups. In tRNA, there is a vast range of modifications, ranging from simple methylation to wholesale restructuring of the purine ring. Modifications occur in all parts of the tRNA molecule There are >50 different types of modified bases in tRNA.

Figure 7.7 shows some of the more common modified bases. Modifications of pyrimidines (C and U) are less complex than those of purines (A and G). In addition to the modifications of the bases themselves, methylation at the 2–O position of the ribose ring also occurs.

The most common modifications of uridine are straightforward. Methylation at position 5 creates ribothymidine (T). The base is the same commonly found in DNA; but here it is attached to ribose, not deoxyribose. In RNA, thymine constitutes an unusual base, originating by modification of U.

Dihydrouridine (D) is generated by the saturation of a double bond, changing the ring structure. Pseudouridine (ψ) interchanges the positions of N and C atoms (see Figure 24.40). And 4-thiouridine has sulfur substituted for oxygen.

The nucleoside inosine is found normally in the cell as an intermediate in the purine biosynthetic pathway. However, it is not incorporated directly into RNA, where instead its existence depends on modification of A to create I. Other modifications of A include the addition of complex groups.

Two complex series of nucleotides depend on modification of G. The Q bases, such as queuosine, have an additional pentenyl ring added via an NH linkage to the methyl group of 7-methylguanosine. The pentenyl ring may carry various further groups. The Y bases, such as wyosine, have an additional ring fused with the purine ring itself; the extra ring carries a long carbon chain, again to which further groups are added in different cases.

The modification reaction usually involves the alteration of, or addition to, existing bases in the tRNA. An exception is the synthesis of Q bases, where a special enzyme exchanges free queuosine with a guanosine residue in the tRNA. The reaction involves breaking and remaking bonds on either side of the nucleoside.

The modified nucleosides are synthesized by specific tRNA-modifying enzymes. The original nucleoside present at each position can be determined either by comparing the sequence of tRNA with that of its gene or (less efficiently) by isolating precursor molecules that lack some or all of the modifications. The sequences of precursors show that different modifications are introduced at different stages during the maturation of tRNA.

Some modifications are constant features of all tRNA molecules—for example, the D residues that give rise to the name of the D arm, and the ψ found in the TψC sequence. On the 3 side of the anticodon there is always a modified purine, although the modification varies widely.

Other modifications are specific for particular tRNAs or groups of tRNAs. For example, wyosine bases are characteristic of tRNA^Phe in bacteria, yeast, and mammals. There are also some species-specific patterns.

The many tRNA-modifying enzymes (~60 in yeast) vary greatly in specificity (for review see Hopper and Phizicky, 2003). In some cases, a single enzyme acts to make a particular modification at a single position. In other cases, an enzyme can modify bases at several different target positions. Some enzymes undertake single reactions with individual tRNAs; others have a range of substrate molecules. The features recognized by the tRNA-modifying enzymes are unknown, but probably involve recognition of structural features surrounding the site of modification. Some modifications require the successive actions of more than one enzyme.

tRNAs are processed from longer precursors

KEY CONCEPTS:

• A mature tRNA is generated by processing a precursor.
• The 5 end is generated by cleavage by the endonuclease RNAase P.
• The 3 end is generated by cleavage followed by trimming of the last few bases, followed by addition of the common terminal trinucleotide sequence CCA.

tRNAs are commonly synthesized as precursor chains with additional material at one or both ends. Figure 7.6 shows that the extra sequences are removed by combinations of endonucleolytic and exonucleolytic activities. One feature that is common to all tRNAs is that the three nucleotides at the 3 terminus, always the triplet sequence CCA, are not coded in the genome, but are added as part of tRNA processing (for review see Hopper and Phizicky, 2003).

The 5 end of tRNA is generated by a cleavage action catalyzed by the enzyme ribonuclease P.

The enzymes that process the 3 end are best characterized in E. coli, where an endonuclease triggers the reaction by cleaving the precursor downstream, and several exonucleases then trim the end by degradation in the 3 -5 direction. The reaction also involves several enzymes in eukaryotes. It generates a tRNA that needs the CCA trinucleotide sequence to be added to the 3 end.

The addition of CCA is the result solely of an enzymatic process, that is, the enzymatic activity carries the specificity for the sequence of the trinucleotide, which is not determined by a template. There are several models for the process, which may be different in different organisms.

In some organisms, the process is catalyzed by a single enzyme. One model for its action proposes that a single enzyme binds to the 3 end, and sequentially adds C, C, and A, the specificity at each stage being determined by the structure of the 3 end. Other models propose that the enzyme has different active sites for CTP and ATP.

In other organisms, different enzymes are responsible for adding the C and A residues, and they function sequentially.

When a tRNA is not properly processed, it attracts the attention of a quality control system that degrades it. This ensures that the protein synthesis apparatus does not become blocked by nonfunctional tRNAs.

Codon-anticodon recognition involves wobbling

KEY TERMS:

• The wobble hypothesis accounts for the ability of a tRNA to recognize more than one codon by unusual (non-G·C, non-A·T) pairing with the third base of a codon.

KEY CONCEPTS:

• Multiple codons that represent the same amino acid most often differ at the third base position.
• The wobble in pairing between the first base of the anticodon and the third base of the codon results from the structure of the anticodon loop. 

The function of tRNA in protein synthesis is fulfilled when it recognizes the codon in the ribosomal A site. The interaction between anticodon and codon takes place by base pairing, but under rules that extend pairing beyond the usual G·C and A·U partnerships.

We can deduce the rules governing the interaction from the sequences of the anticodons that correspond to particular codons. The ability of any tRNA to respond to a given codon can be measured directly by the trinucleotide binding assay or by its use in an in vitro protein synthetic system.

The genetic code itself yields some important clues about the process of codon recognition. The pattern of third-base degeneracy is drawn in Figure 7.3, which shows that in almost all cases either the third base is irrelevant or a distinction is made only between purines and pyrimidines.

There are eight codon families in which all four codons sharing the same first two bases have the same meaning, so that the third base has no role at all in specifying the amino acid. There are seven codon pairs in which the meaning is the same whichever pyrimidine is present at the third position; and there are five codon pairs in which either purine may be present without changing the amino acid that is coded.

There are only three cases in which a unique meaning is conferred by the presence of a particular base at the third position: AUG (for methionine), UGG (for tryptophan), and UGA (termination). So C and U never have a unique meaning in the third position, and A never signifies a unique amino acid.

Because the anticodon is complementary to the codon, it is the first base in the anticodon sequence written conventionally in the direction from 5 to 3 that pairs with the third base in the codon sequence written by the same convention. So the combination

Codon     5 A C G 3

Anticodon 3 U G C 5

is usually written as codon ACG/anticodon CGU, where the anticodon sequence must be read backward for complementarity with the codon.

To avoid confusion, we shall retain the usual convention in which all sequences are written 5–3, but indicate anticodon sequences with a backward arrow as a reminder of the relationship with the codon. So the codon/anticodon pair shown above will be written as ACG and CGU, respectively.

Does each triplet codon demand its own tRNA with a complementary anticodon? Or can a single tRNA respond to both members of a codon pair and to all (or at least some) of the four members of a codon family?

Often one tRNA can recognize more than one codon. This means that the base in the first position of the anticodon must be able to partner alternative bases in the corresponding third position of the codon. Base pairing at this position cannot be limited to the usual G·C and A·U partnerships.

The rules governing the recognition patterns are summarized in the wobble hypothesis, which states that the pairing between codon and anticodon at the first two codon positions always follows the usual rules, but that exceptional "wobbles" occur at the third position. Wobbling occurs because the conformation of the tRNA anticodon loop permits flexibility at the first base of the anticodon (Crick, 1966). Figure 7.4 shows that G·U pairs can form in addition to the usual pairs.

This single change creates a pattern of base pairing in which A can no longer have a unique meaning in the codon (because the U that recognizes it must also recognize G). Similarly, C also no longer has a unique meaning (because the G that recognizes it also must recognize U). Figure 7.5 summarizes the pattern of recognition.

It is therefore possible to recognize unique codons only when the third bases are G or U; this option is not used often, since UGG and AUG are the only examples of the first type, and there is none of the second type.

(G·U pairs are common in RNA duplex structures. But the formation of stable contacts between codon and anticodon, when only 3 base pairs can be formed, is more constrained, and thus G·U pairs can contribute only in the last position of the codon.)

Related codons represent related amino acids

KEY TERMS:

• Synonym codons have the same meaning in the genetic code. Synonym tRNAs bear the same amino acid and respond to the same codon.
• Third base degeneracy describes the lesser effect on codon meaning of the nucleotide present in the third codon position.
• A stop codon (Termination codon) is one of three triplets (UAG, UAA, UGA) that causes protein synthesis to terminate. They are also known historically as nonsense codons. The UAA codon is called ochre, and the UAA codon is called amber, after the names of the nonsense mutations by which they were originally identified.

KEY CONCEPTS:

• 61 of the 64 possible triplets code for 20 amino acids.
• 3 codons do not represent amino acids and cause termination.
• The genetic code was frozen at an early stage of evolution and is universal.
• Most amino acids are represented by more than one codon.
• The multiple codons for an amino acid are usually related.
• Related amino acids often have related codons, minimizing the effects of mutation. 

The code is summarized in Figure 7.1. Because there are more codons (61) than there are amino acids (20), almost all amino acids are represented by more than one codon. The only exceptions are methionine and tryptophan. Codons that have the same meaning are called synonyms. Because the genetic code is actually read on the mRNA, usually it is described in terms of the four bases present in RNA: U, C, A, and G.

Codons representing the same or related amino acids tend to be similar in sequence. Often the base in the third position of a codon is not significant, because the four codons differing only in the third base represent the same amino acid. Sometimes a distinction is made only between a purine versus a pyrimidine in this position. The reduced specificity at the last position is known as third base degeneracy.

The interpretation of a codon requires base pairing with the anticodon of the corresponding aminoacyl-tRNA. The reaction occurs within the ribosome: complementary trinucleotides in isolation would usually be too short to pair in a stable manner, but the interaction is stabilized by the environment of the ribosomal A site. Also, base pairing between codon and anticodon is not solely a matter of A·U and G·C base pairing. The ribosome controls the environment in such a way that conventional pairing occurs at the first two positions of the codon, but additional reactions are permitted at the third base. As a result, a single aminoacyl-tRNA may recognize more than one codon, corresponding with the pattern of degeneracy. Furthermore, pairing interactions may also be influenced by the introduction of special bases into tRNA, especially by modification in or close to the anticodon.

The tendency for similar amino acids to be represented by related codons minimizes the effects of mutations. It increases the probability that a single random base change will result in no amino acid substitution or in one involving amino acids of similar character. For example, a mutation of CUC to CUG has no effect, since both codons represent leucine; and a mutation of CUU to AUU results in replacement of leucine with isoleucine, a closely related amino acid.

Figure 7.2 plots the number of codons representing each amino acid against the frequency with which the amino acid is used in proteins (in E. coli). There is only a slight tendency for amino acids that are more common to be represented by more codons, and therefore it does not seem that the genetic code has been optimized with regard to the utilization of amino acids.

The three codons (UAA, UAG, and UGA) that do not represent amino acids are used specifically to terminate protein synthesis. One of these stop codons marks the end of every gene.

Is the genetic code the same in all living organisms?

Comparisons of DNA sequences with the corresponding protein sequences reveal that the identical set of codon assignments is used in bacteria and in eukaryotic cytoplasm. As a result, mRNA from one species usually can be translated correctly in vitro or in vivo by the protein synthetic apparatus of another species. So the codons used in the mRNA of one species have the same meaning for the ribosomes and tRNAs of other species.

The universality of the code argues that it must have been established very early in evolution. Perhaps the code started in a primitive form in which a small number of codons were used to represent comparatively few amino acids, possibly even with one codon corresponding to any member of a group of amino acids. More precise codon meanings and additional amino acids could have been introduced later. One possibility is that at first only two of the three bases in each codon were used; discrimination at the third position could have evolved later. (Originally there might have been a stereochemical relationship between amino acids and the codons representing them. Then a more complex system evolved.)

Evolution of the code could have become "frozen" at a point at which the system had become so complex that any changes in codon meaning would disrupt existing proteins by substituting unacceptable amino acids. Its universality implies that this must have happened at such an early stage that all living organisms are descended from a single pool of primitive cells in which this occurred.

Exceptions to the universal genetic code are rare. Changes in meaning in the principal genome of a species usually concern the termination codons. For example, in a mycoplasma, UGA codes for tryptophan; and in certain species of the ciliates Tetrahymena and Paramecium, UAA and UAG code for glutamine. Systematic alterations of the code have occurred only in mitochondrial DNA (see 7.7 There are sporadic alterations of the universal code). 

USING THE GENETIC CODE

The sequence of a coding strand of DNA, read in the direction from 5 to 3, consists of nucleotide triplets (codons) corresponding to the amino acid sequence of a protein read from N-terminus to C-terminus. Sequencing of DNA and proteins makes it possible to compare corresponding nucleotide and amino acid sequences directly. There are 64 codons (each of 4 possible nucleotides can occupy each of the three positions of the codon, making 4³ = 64 possible trinucleotide sequences). Each of these codons has a specific meaning in protein synthesis: 61 codons represent amino acids; 3 codons cause the termination of protein synthesis.

The meaning of a codon that represents an amino acid is determined by the tRNA that corresponds to it; the meaning of the termination codons is determined directly by protein factors.

The breaking of the genetic code originally showed that genetic information is stored in the form of nucleotide triplets, but did not reveal how each codon specifies its corresponding amino acid. Before the advent of sequencing, codon assignments were deduced on the basis of two types of in vitro studies. A system involving the translation of synthetic polynucleotides was introduced in 1961, when Nirenberg showed that polyuridylic acid [poly(U)] directs the assembly of phenylalanine into polyphenylalanine. This result means that UUU must be a codon for phenylalanine. A second system was later introduced in which a trinucleotide was used to mimic a codon, thus causing the corresponding aminoacyl-tRNA to bind to a ribosome. By identifying the amino acid component of the aminoacyl-tRNA, the meaning of the codon can be found. The two techniques together assigned meaning to all of the codons that represent amino acids (Nirenberg and Matthaei, 1961; Nirenberg and Leder, 1964).

61 of the 64 codons represent amino acids. The other three cause termination of protein synthesis. The assignment of amino acids to codons is not random, but shows relationships in which the third base has less effect on codon meaning; also related amino acids are often represented by related codons.

23S rRNA has peptidyl transferase activity

KEY CONCEPTS:

• Peptidyl transferase activity resides exclusively in the 23S rRNA. 

The sites involved in the functions of 23S rRNA are less well identified than those of 16S rRNA, but the same general pattern is observed: bases at certain positions affect specific functions. Bases at some positions in 23S rRNA are affected by the conformation of the A site or P site. In particular, oligonucleotides derived from the 3 CCA terminus of tRNA protect a set of bases in 23S rRNA which essentially are the same as those protected by peptidyl-tRNA. This suggests that the major interaction of 23S rRNA with peptidyl-tRNA in the P site involves the 3 end of the tRNA.

The tRNA makes contacts with the 23S rRNA in both the P and A sites. At the P site, G2552 of 23S rRNA base pairs with C74 of the peptidyl tRNA. A mutation in the G in the rRNA prevents interaction with tRNA, but interaction is restored by a compensating mutation in the C of the amino acceptor end of the tRNA. At the A site, G2553 of the 23S rRNA base pairs with C75 of the aminoacyl-tRNA. So there is a close role for rRNA in both the tRNA-binding sites. Indeed, when we have a clearer structural view of the region, we should be able to understand the movements of tRNA between the A and P sites in terms of making and breaking contacts with rRNA (Samaha, Green, and Noller, 1995).

Another site that binds tRNA is the E site, which is localized almost exclusively on the 50S subunit. Bases affected by its conformation can be identified in 23S rRNA.

What is the nature of the site on the 50S subunit that provides peptidyl transferase function? The involvement of rRNA was first indicated because a region of the 23S rRNA is the site of mutations that confer resistance to antibiotics that inhibit peptidyl transferase.

A long search for ribosomal proteins that might possess the catalytic activity has been unsuccessful. More recent results suggest that the ribosomal RNA of the large subunit has the catalytic activity. Extraction of almost all the protein content of 50S subunits leaves the 23S rRNA associated largely with fragments of proteins, amounting to <5% of the mass of the ribosomal proteins. This preparation retains peptidyl transferase activity. Treatments that damage the RNA abolish the catalytic activity (Noller, Hoffarth, and Zimniak, 1992).

Following from these results, 23S rRNA prepared by transcription in vitro can catalyze the formation of a peptide bond between Ac-Phe-tRNA and Phe-tRNA. The yield of Ac-Phe-Phe is very low, suggesting that the 23S rRNA requires proteins in order to function at a high efficiency. But since the rRNA has the basic catalytic activity, the role of the proteins must be indirect, serving to fold the rRNA properly or to present the substrates to it. The reaction also works, although less effectively, if the domains of 23S rRNA are synthesized separately and then combined. In fact, some activity is shown by domain V alone, which has the catalytic center. Activity is abolished by mutations in position 2252 of domain V that lies in the P site.

The crystal structure of an archaeal 50S subunit shows that the peptidyl transferase site basically consists of 23S rRNA (Ban et al., 2000). There is no protein within 18Å of the active site where the transfer reaction occurs between peptidyl-tRNA and aminoacyl-tRNA!

Peptide bond synthesis requires an attack by the amino group of one amino acid on the carboxyl group of another amino acid. Catalysis requires a basic residue to accept the hydrogen atom that is released from the amino group, as shown in Figure 6.48. If rRNA is the catalyst, it must provide this residue, but we do not know how this happens. The purine and pyrimidine bases are not basic at physiological pH. A highly conserved base (at position 2451 in E. coli) had been implicated, but appears now neither to have the right properties nor to be crucial for peptidyl transferase activity (Bayfield et al., 2001; Thompson et al., 2001).

Proteins that are bound to the 23S rRNA outside of the peptidyl transfer region are almost certainly required to enable the rRNA to form the proper structure in vivo. The idea that rRNA is the catalytic component is consistent with the results discussed in 24 RNA splicing and processingthat identify catalytic properties in RNA that are involved with several RNA processing reactions. It fits with the notion that the ribosome evolved from functions originally possessed by RNA.

..

16S rRNA plays an active role in protein synthesis

KEY CONCEPTS:

• 16S rRNA plays an active role in the functions of the 30S subunit. It interacts directly with mRNA, with the 50S subunit, and with the anticodons of tRNAs in the P and A sites. 

The ribosome was originally viewed as a collection of proteins with various catalytic activities, held together by protein-protein interactions and by binding to rRNA. But the discovery of RNA molecules with catalytic activities (see 24 RNA splicing and processing) immediately suggests that rRNA might play a more active role in ribosome function. There is now evidence that rRNA interacts with mRNA or tRNA at each stage of translation, and that the proteins are necessary to maintain the rRNA in a structure in which it can perform the catalytic functions. Several interactions involve specific regions of rRNA:

• The 3 terminus of the rRNA interacts directly with mRNA at initiation.
• Specific regions of 16S rRNA interact directly with the anticodon regions of tRNAs in both the A site and the P site. Similarly, 23S rRNA interacts with the CCA terminus of peptidyl-tRNA in both the P site and A site.
• Subunit interaction involves interactions between 16S and 23S rRNAs (see 6.16 Ribosomal RNA pervades both ribosomal subunits).

Much information about the individual steps of bacterial protein synthesis has been obtained by using antibiotics that inhibit the process at particular stages. The target for the antibiotic can be identified by the component in which resistant mutations occur. Some antibiotics act on individual ribosomal proteins, but several act on rRNA, which suggests that the rRNA is involved with many or even all of the functions of the ribosome.

The functions of rRNA have been investigated by two types of approach. Structural studies show that particular regions of rRNA are located in important sites of the ribosome, and that chemical modifications of these bases impede particular ribosomal functions. And mutations identify bases in rRNA that are required for particular ribosomal functions. Figure 6.45 summarizes the sites in 16S rRNA that have been identified by these means (for review see Noller, 1991).

An indication of the importance of the 3 end of 16S rRNA is given by its susceptibility to the lethal agent colicin E3. Produced by some bacteria, the colicin cleaves ~50 nucleotides from the 3 end of the 16S rRNA of E. coli. The cleavage entirely abolishes initiation of protein synthesis. Several important functions require the region that is cleaved: binding the factor IF-3; recognition of mRNA; and binding of tRNA.

The 3 end of the 16S rRNA is directly involved in the initiation reaction by pairing with the Shine-Dalgarno sequence in the ribosome-binding site of mRNA (see Figure 6.16 and Great Experiments:  rRNA-mRNA base pairing selects translational initiator regions in bacteria). Another direct role for the 3 end of 16S rRNA in protein synthesis is shown by the properties of kasugamycin-resistant mutants, which lack certain modifications in 16S rRNA. Kasugamycin blocks initiation of protein synthesis. Resistant mutants of the type ksgA lack a methylase enzyme that introduces four methyl groups into two adjacent adenines at a site near the 3 terminus of the 16S rRNA. The methylation generates the highly conserved sequence G–m₂⁶A–m₂⁶A, found in both prokaryotic and eukaryotic small rRNA. The methylated sequence is involved in the joining of the 30S and 50S subunits, which in turn is connected also with the retention of initiator tRNA in the complete ribosome. Kasugamycin causes fMet-tRNA_f to be released from the sensitive (methylated) ribosomes, but the resistant ribosomes are able to retain the initiator.

Changes in the structure of 16S rRNA occur when ribosomes are engaged in protein synthesis, as seen by protection of particular bases against chemical attack. The individual sites fall into a few groups, concentrated in the 3 minor and central domains. Although the locations are dispersed in the linear sequence of 16S rRNA, it seems likely that base positions involved in the same function are actually close together in the tertiary structure.

Some of the changes in 16S rRNA are triggered by joining with 50S subunits, binding of mRNA, or binding of tRNA. They indicate that these events are associated with changes in ribosome conformation that affect the exposure of rRNA. They do not necessarily indicate direct participation of rRNA in these functions. One change that occurs during protein synthesis is shown in Figure 6.46; it involves a local movement to change the nature of a short duplex sequence.

The 16S rRNA is involved in both A site and P site function, and significant changes in its structure occur when these sites are occupied (Moazed and Noller, 1986). Certain distinct regions are protected by tRNA bound in the A site (see Figure 6.45). One is the 530 loop (which is also is the site of a mutation that prevents termination at the UAA, UAG, and UGA codons). The other is the 1400-1500 region (so-called because bases 1399-1492 and the adenines at 1492-1493 are two single-stranded stretches that are connected by a long hairpin). All of the effects that tRNA binding has on 16S rRNA can be produced by the isolated oligonucleotide of the anticodon stem-loop, so that tRNA-30S subunit binding must involve this region (Lodmell and Dahlberg, 1997).

The adenines at 1492-1493 provide a mechanism for detecting properly paired codon-anticodon complexes (Yoshizawa, Fourmy, and Puglisi, 1999). The principle of the interaction is that the structure of the 16S rRNA responds to the structure of the first two bases pairs in the minor groove of the duplex formed by the codon-anticodon interaction. Modification of the N1 position of either base 1492 or 1493 in rRNA prevents tRNA from binding in the A site. However, mutations at 1492 or 1493 can be suppressed by the introduction of fluorine at the 2 position of the corresponding bases in mRNA (which restores the interaction). Figure 6.47 shows that codon-anticodon pairing allows the N1 of each adenine to interact with the 2–OH in the mRNA backbone. When an incorrect tRNA enters the A site, the structure of the codon-anticodon complex is distorted, and this interaction cannot occur. The interaction stabilizes the association of tRNA with the A site.

A variety of bases in different positions of 16S rRNA are protected by tRNA in the P site; probably the bases lie near one another in the tertiary structure. In fact, there are more contacts with tRNA when it is in the P site than when it is in the A site. This may be responsible for the increased stability of peptidyl-tRNA compared with aminoacyl-tRNA. This makes sense, because once the tRNA has reached the P site, the ribosome has decided that it is correctly bound, whereas in the A site, the assessment of binding is being made. The 1400 region can be directly cross-linked to peptidyl-tRNA, which suggests that this region is a structural component of the P site.

The basic conclusion to be drawn from these results is that rRNA has many interactions with both tRNA and mRNA, and that these interactions recur in each cycle of peptide bond formation.