October 19, 2012

Anchor sequences determine protein orientation

KEY TERMS:
  • An anchor (stop-transfer) (often referred to as a "transmembrane anchor") is a segment of a transmembrane protein which resides in the membrane.
KEY CONCEPTS:
  • An anchor sequence halts the passage of a protein through the translocon. Typically this is located at the C-terminal end and results in a group I orientation in which the N-terminus has passed through the membrane.
  • A combined signal-anchor sequence can be used to insert a protein into the membrane and anchor the site of insertion. Typically this is internal and results in a group II orientation in which the N-terminus is cytosolic. 

October 18, 2012

Proteins reside in membranes by means of hydrophobic regions

KEY TERMS:
  • The transmembrane region (transmembrane domain) is the part of a protein that spans the membrane bilayer. It is hydrophobic and in many cases contains approximately 20 amino acids that form an α-helix. It is also called the transmembrane domain.
  • A transmembrane protein (Integral membrane protein) extends across a lipid bilayer. A hydrophobic region (typically consisting of a stretch of 20-25 hydrophobic and/or uncharged aminoa acids) or regions of the protein resides in the membrane. Hydrophilic regions are exposed on one or both sides of the membrane.

Reverse translocation sends proteins to the cytosol for degradation

KEY TERMS:
  • Retrograde translocation (Reverse translocation) is the translocation of a protein from the lumen of the ER to the cytoplasm. It usually occurs to allow misfolded or damaged proteins to be degraded by the proteasome.
KEY CONCEPTS:
  • Sec61 translocons can be used for reverse translocation of proteins from the ER into the cytosol. 

Translocation requires insertion into the translocon and (sometimes) a ratchet in the ER

KEY CONCEPTS:
  • The ribosome, SRP, and SRP receptor are sufficient to insert a nascent protein into a translocon.
  • Proteins that are inserted post-translationally require additional components in the cytosol and Bip in the ER.
  • Bip is a ratchet that prevents a protein from slipping backward.
The translocon and the SRP receptor are the basic components required for co-translational translocation. When the Sec61 complex is incorporated into artificial membranes together with the SRP receptor, it can support translocation of some nascent proteins. Other nascent proteins require the presence of an additional component, TRAM, which is a major protein that becomes crosslinked to a translocating nascent chain. TRAM stimulates the translocation of all proteins.

The translocon forms a pore

KEY TERMS:
  • A translocon is a discrete structure in a membrane that forms a channel through which (hydrophilic) proteins may pass.
KEY CONCEPTS:
  • The Sec61 trimeric complex provides the channel for proteins to pass through a membrane.
  • A translocating protein passes directly from the ribosome to the translocon without exposure to the cytosol. 

The SRP interacts with the SRP receptor

KEY TERMS:
  • The Alu domain comprises the parts of the 7S RNA of the SRP that are related to Alu RNA.
  • The S domain is the sequence of 7S RNA of the SRP that is not related to Alu RNA.
KEY CONCEPTS:
  • The SRP is a complex of 7S RNA with 6 proteins.
  • The bacterial equivalent to the SRP is a complex of 4.5S RNA with two proteins.
  • The SRP receptor is a dimer.
  • GTP hydrolysis releases the SRP from the SRP receptor after their interaction. 

The signal sequence interacts with the SRP

KEY TERMS:
  • Protein translocation describes the movement of a protein across a membrane. This occurs across the membranes of organelles in eukaryotes, or across the plasma membrane in bacteria. Each membrane across which proteins are translocated has a channel specialized for the purpose.
  • The signal recognition particle (SRP) is a ribonucleoprotein complex that recognizes signal sequences during translation and guides the ribosome to the translocation channel. SRPs from different organisms may have different compositions, but all contain related proteins and RNAs.
  • Signal peptidase is an enzyme within the membrane of the ER that specifically removes the signal sequences from proteins as they are translocated. Analogous activities are present in bacteria, archaebacteria, and in each organelle in a eukaryotic cell into which proteins are targeted and translocated by means of removable targeting sequences. Signal peptidase is one component of a larger protein complex.

Signal sequences initiate translocation


KEY TERMS:
  • Protein sorting (targeting) is the direction of different types of proteins for transport into or between specific organelles.
  • A signal sequence is a short region of a protein that directs it to the endoplasmic reticulum for co-translational translocation.
KEY CONCEPTS:
  • Proteins associate with the ER system only co-translationally.
  • The signal sequence of the substrate protein is responsible for membrane association. 

Proteins that associate with membranes via N-terminal leaders use a hierarchy of signals to find their final destination. In the case of the reticuloendothelial system, the ultimate location of a protein depends on how it is directed as it transits the endoplasmic reticulum and Golgi apparatus. The leader sequence itself introduces the protein to the membrane; the intrinsic consequence of the interaction is for the protein to pass through the membrane into the compartment on the other side. For a protein to reside within the membrane, a further signal is required to stop passage through the membrane. Other types of signals are required for a protein to be sorted to a particular destination, that is, to remain within the membrane or lumen of some particular compartment. The general process of finding its ultimate destination by transport through successive membrane systems is called protein sorting or targeting, and is discussed in 27 Protein trafficking.

The overall nature of the pathway is summarized in Figure 8.18. The "default pathway" takes a protein through the ER, into the Golgi, and on to the plasma membrane. Proteins that reside in the ER possess a C-terminal tetrapeptide (KDEL, which actually provides a signal for them to return to the ER from the Golgi). The signal that diverts a protein to the lysosome is a covalent modification: the addition of a particular sugar residue. Other signals are required for a protein to become a permanent constituent of the Golgi or the plasma membrane. We discuss direction to these locations in 27 Protein trafficking.






There is a common starting point for proteins that associate with, or pass through, the reticuloendothelial system of membranes. These proteins can associate with the membrane only while they are being synthesized. The ribosomes synthesizing these proteins become associated with the endoplasmic reticulum, enabling the nascent protein to be co-translationally transferred to the membrane. Regions in which ribosomes are associated with the ER are sometimes called the "rough ER," in contrast with the "smooth ER" regions that lack associated polysomes and which have a tubular rather than sheet-like appearance (for review see Palade, 1975). Figure 8.19 shows ribosomes in the act of transferring nascent proteins to ER membranes.
The proteins synthesized at the rough endoplasmic reticulum pass from the ribosome directly to the membrane. Then they are transferred to the Golgi apparatus, and finally are directed to their ultimate destination, such as the lysosome or secretory vesicle or plasma membrane. The process occurs within a membranous environment as the proteins are carried between organelles in small membrane-coated vesicles (see 27 Protein trafficking.)

Co-translational insertion is directed by a signal sequence. Usually this is a cleavable leader sequence of 15-30 N-terminal amino acids. At or close to the N-terminus are several polar residues, and within the leader is a hydrophobic core consisting exclusively or very largely of hydrophobic amino acids (for review see von Heijne, 1985). There is no other conservation of sequence. Figure 8.20 gives an example.
The signal sequence is both necessary and sufficient to sponsor transfer of any attached polypeptide into the target membrane. A signal sequence added to the N-terminus of a globin protein, for example, causes it to be secreted through cellular membranes instead of remaining in the cytosol (Lingappa et al., 1984).
The signal sequence provides the connection that enables the ribosomes to attach to the membrane. There is no intrinsic difference between free ribosomes (synthesizing proteins in the cytosol) and ribosomes that are attached to the ER. A ribosome starts synthesis of a protein without knowing whether the protein will be synthesized in the cytosol or transferred to a membrane. It is the synthesis of a signal sequence that causes the ribosome to associate with a membrane (Blobel and Dobberstein, 1975; for review see Lee and Beckwith, 1986).

October 16, 2012

Hsp60/GroEL forms an oligomeric ring structure

KEY CONCEPTS:
  • Hsp60/GroEL forms an oligomeric structure consisting of 14 subunits arranged in two inverted heptameric rings.
  • A GroES heptamer forms a dome that caps one end of the double ring.
  • A substrate protein undergoes a cycle of folding in the cavity of one of the Hsp60/GroEL rings. It is released and rebound for further cycles until it reaches mature conformation.
  • Hydrolysis of ATP provides energy for the folding cycles.

The Hsp70 family is ubiquitous

KEY CONCEPTS:
  • Hsp70 is a chaperone that functions on target proteins in conjunction with DnaJ and GrpE.
  • Members of the Hsp70 family are found in the cytosol, in the ER, and in mitochondria and chloroplasts.
The Hsp70 family is found in bacteria, eukaryotic cytosol, in the endoplasmic reticulum, and in chloroplasts and mitochondria (for review see Frydman, 2001). A typical Hsp70 has two domains: the N-terminal domain is an ATPase; and the C-terminal domain binds the substrate polypeptide (Flaherty, DeLuca-Flaherty, and McKay, 1990; Zhu et al., 1996). When bound to ATP, Hsp70 binds and releases substrates rapidly; when bound to ADP, the reactions are slow. Recycling between these states is regulated by two other proteins, Hsp40 (DnaJ) and GrpE.

Chaperones are needed by newly synthesized and by denatured proteins

KEY CONCEPTS:
  • Chaperones act on newly synthesized proteins, proteins that are passing through membranes, or proteins that have been denatured.
  • Hsp70 and some associated proteins form a major class of chaperones that act on many target proteins.
  • Group I and group II chaperonins are large oligomeric assemblies that act on target proteins they sequester in internal cavities.
  • Hsp90 is a specialized chaperone that acts on proteins of signal transduction pathways. 

Chaperones may be required for protein folding

KEY TERMS:
  • Self-assembly refers to the ability of a protein (or of a complex of proteins) to form its final structure without the intervention of any additional components (such as chaperones). The term can also refer to the spontaneous formation of any biological structure that occurs when molecules collide and bind to each other.
  • Chaperones are a class of proteins which bind to incompletely folded or assembled proteins in order to assist their folding or prevent them from aggregating.

Protein translocation may be post-translational or co-translational

KEY TERMS:
  • The leader of a protein is a short N-terminal sequence responsible for initiating passage into or through a membrane.
  • A protein to be imported into an organelle or secreted from bacteria is called a "preprotein" until its signal sequence has been removed.

Passage across a membrane requires a special apparatus

KEY TERMS:
  • Protein translocation describes the movement of a protein across a membrane. This occurs across the membranes of organelles in eukaryotes, or across the plasma membrane in bacteria. Each membrane across which proteins are translocated has a channel specialized for the purpose.
KEY CONCEPTS:
  • Proteins pass across membranes through specialized protein structures embedded in the membrane.
  • Substrate proteins interact directly with the transport apparatus of the ER or mitochondria or chloroplasts, but require carrier proteins to interact with peroxisomes.
  • A much larger and complex apparatus is required for transport into the nucleus. 

PROTEIN LOCALIZATION


Proteins are synthesized in two types of location:
  • The vast majority of proteins are synthesized by ribosomes in the cytosol.
  • A small minority are synthesized by ribosomes within organelles (mitochondria or chloroplasts).
Proteins synthesized in the cytosol can be divided into two general classes with regard to localization: those that are not associated with membranes; and those that are associated with membranes (see 32.5 Membranes and membrane proteins). Figure 8.1 maps the cell in terms of the possible ultimate destinations for a newly synthesized protein and the systems that transport it:

Bypassing involves ribosome movement


Certain sequences trigger a bypass event, when a ribosome stops translation, slides along mRNA with peptidyl-tRNA remaining in the P site, and then resumes translation (see Figure 7.30). This is a rather rare phenomenon, with only ~3 authenticated examples (for review see Herr, Atkins, and Gesteland, 2000). The most dramatic example of bypassing is in gene 60 of phage T4, where the ribosome moves 60 nucleotides along the mRNA (Huang et al., 1988).

The key to the bypass system is that there are identical (or synonymous) codons at either end of the sequence that is skipped. They are sometimes referred to as the "take-off" and "landing" sites. Before bypass, the ribosome is positioned with a peptidyl-tRNA paired with the take-off codon in the P site, with an empty A site waiting for an aminoacyl-tRNA to enter. Figure 7.32 shows that the ribosome slides along mRNA in this condition until the peptidyl-tRNA can become paired with the codon in the landing site. A remarkable feature of the system is its high efficiency, ~50%.
The sequence of the mRNA triggers the bypass. The important features are the two GGA codons for take-off and landing, the spacing between them, a stem-loop structure that includes the take-off codon, and the stop codon adjacent to the take-off codon. The protein under synthesis is also involved.
The take-off stage requires the peptidyl-tRNA to unpair from its codon. This is followed by a movement of the mRNA that prevents it from re-pairing. Then the ribosome scans the mRNA until the peptidyl-tRNA can repair with the codon in the landing reaction. This is followed by the resumption of protein synthesis when aminoacyl-tRNA enters the A site in the usual way.
Like frameshifting, the bypass reaction depends on a pause by the ribosome. The probability that peptidyl-tRNA will dissociate from its codon in the P site is increased by delays in the entry of aminoacyl-tRNA into the A site. Starvation for an amino acid can trigger bypassing in bacterial genes because of the delay that occurs when there is no aminoacyl-tRNA available to enter the A site (Gallant and Lindsley, 1998). In phage T4 gene 60, one role of mRNA structure may be to reduce the efficiency of termination, thus creating the delay that is needed for the take-off reaction.  

Frameshifting occurs at slippery sequences


KEY TERMS:
  • Programmed frameshifting is required for expression of the protein sequences coded beyond a specific site at which a +1 or -1 frameshift occurs at some typical frequency.
KEY CONCEPTS:
  • The reading frame may be influenced by the sequence of mRNA and the ribosomal environment.
  • Slippery sequences allow a tRNA to shift by 1 base after it has paired with its anticodon, thereby changing the reading frame.
  • Translation of some genes depends upon the regular occurrence of programmed frameshifting. 

Frameshifting is associated with specific tRNAs in two circumstances (for review see Farabaugh and Bjorkk, 1999):
  • Some mutant tRNA suppressors recognize a "codon" for 4 bases instead of the usual 3 bases.
  • Certain "slippery" sequences allow a tRNA to move a base up or down mRNA in the A site.
Frameshift mutants result from the insertion or deletion of a base. They can be suppressed by restoring the original reading frame. This can be achieved by compensating base deletions and insertions within a gene (see 1.21 The genetic code is triplet). However, extragenic frameshift suppressors also can be found in the form of tRNAs with aberrant properties.
The simplest type of external frameshift suppressor corrects the reading frame when a mutation has been caused by inserting an additional base within a stretch of identical residues. For example, a G may be inserted in a run of several contiguous G bases. The frameshift suppressor is a tRNAGly that has an extra base inserted in its anticodon loop, converting the anticodon from the usual triplet sequence CCC to the quadruplet sequence CCCC . The suppressor tRNA recognizes a 4-base "codon".
Some frameshift suppressors can recognize more than one 4-base "codon". For example, a bacterial tRNALys suppressor can respond to either AAAA or AAAU, instead of the usual codon AAA. Another suppressor can read any 4-base "codon" with ACC in the first three positions; the next base is irrelevant. In these cases, the alternative bases that are acceptable in the fourth position of the longer "codon" are not related by the usual wobble rules. The suppressor tRNA probably recognizes a 3 base codon, but for some other reasonmost likely steric hindrancethe adjacent base is blocked. This forces one base to be skipped before the next tRNA can find a codon.
Situations in which frameshifting is a normal event are presented by phages and viruses. Such events may affect the continuation or termination of protein synthesis, and result from the intrinsic properties of the mRNA.

In retroviruses, translation of the first gene is terminated by a nonsense codon in phase with the reading frame. The second gene lies in a different reading frame, and (in some viruses) is translated by a frameshift that changes into the second reading frame and therefore bypasses the termination codon (see Figure 7.29) (Jacks et al., 1988) (see 17.3 Retroviral genes codes for polyproteins). The efficiency of the frameshift is low, typically ~5%. In fact, this is important in the biology of the virus; an increase in efficiency can be damaging. Figure 7.31 illustrates the similar situation of the yeast Ty element, in which the termination codon of tya must be bypassed by a frameshift in order to read the subsequent tyb gene.
Such situations makes the important point that the rare (but predictable) occurrence of "misreading" events can be relied on as a necessary step in natural translation. This is called programmed frameshifting (for review see Farabaugh, 1995; Gesteland and Atkins, 1996). It occurs at particular sites at frequencies that are 100-1000× greater than the rate at which errors are made at nonprogrammed sites (~3 × 105 per codon).
There are two common features in this type of frameshifting:
  • A "slippery" sequence allows an aminoacyl-tRNA to pair with its codon and then to move +1 (rare) or 1 base (more common) to pair with an overlapping triplet sequence that can also pair with its anticodon.
  • The ribosome is delayed at the frameshifting site to allow time for the aminoacyl-tRNA to rearrange its pairing. The cause of the delay can be an adjacent codon that requires a scarce aminoacyl-tRNA, a termination codon that is recognized slowly by its release factor, or a structural impediment in mRNA (for example, a "pseudoknot," a particular conformation of RNA) that impedes the ribosome.
Slippery events can involve movement in either direction; a 1 frameshift is caused when the tRNA moves backwards, and a +1 frameshift is caused when it moves forwards. In either case, the result is to expose an out-of-phase triplet in the A site for the next aminoacyl-tRNA. The frameshifting event occurs before peptide bond synthesis. In the most common type of case, when it is triggered by a slippery sequence in conjunction with a downstream hairpin in mRNA, the surrounding sequences influence its efficiency.
The frameshifting in Figure 7.31 shows the behavior of a typical slippery sequence. The 7 nucleotide sequence CUUAGGC is usually recognized by Leu-tRNA at CUU followed by Arg-tRNA at AGC. However, the Arg-tRNA is scarce, and when its scarcity results in a delay, the Leu-tRNA slips from the CUU codon to the overlapping UUA triplet. This causes a frameshift, because the next triplet in phase with the new pairing (GGC) is read by Gly-tRNA. Slippage usually occurs in the P site (when the Leu-tRNA actually has become peptidyl-tRNA, carrying the nascent chain).
Frameshifting at a stop codon causes readthrough of the protein. The base on the 3 side of the stop codon influences the relative frequencies of termination and frameshifting, and thus affects the efficiency of the termination signal. This helps to explain the significance of context on termination.

Recoding changes codon meanings


KEY TERMS:
  • Recoding events occur when the meaning of a codon or series of codons is changed from that predicted by the genetic code. It may involve altered interactions between aminoacyl-tRNA and mRNA that are influenced by the ribosome.
KEY CONCEPTS:
  • Changes in codon meaning can be caused by mutant tRNAs or by tRNAs with special properties.
  • The reading frame can be changed by frameshifting or bypassing, both of which depend on properties of the mRNA. 

The reading frame of a messenger usually is invariant. Translation starts at an AUG codon and continues in triplets to a termination codon. Reading takes no notice of sense: insertion or deletion of a base causes a frameshift mutation, in which the reading frame is changed beyond the site of mutation. Ribosomes and tRNAs continue ineluctably in triplets, synthesizing an entirely different series of amino acids.
There are some exceptions to the usual pattern of translation that enable a reading frame with an interruption of some sortsuch as a nonsense codon or frameshiftto be translated into a full-length protein. Recoding events are responsible for making exceptions to the usual rules, and can involve several types of events.

Changing the meaning of a single codon allows one amino acid to be substituted in place of another, or for an amino acid to be inserted at a termination codon. Figure 7.28 shows that these changes rely on the properties of an individual tRNA that responds to the codon:
Changing the reading frame occurs in two types of situation:

  • Frameshifting typically involves changing the reading frame when aminoacyl-tRNA slips by one base (+1 forward or 1 backward) (see 7.17 Frameshifting occurs at slippery sequences). The result shown in Figure 7.29 is that translation continues past a termination codon.
  • Bypassing involves a movement of the ribosome to change the codon that is paired with the peptidyl-tRNA in the P site. The sequence between the two codons fails to be represented in protein. As shown in Figure 7.30, this allows translation to continue past any termination codons in the intervening region.








The ribosome influences the accuracy of translation


KEY CONCEPTS:
  • The structure of the 16S rRNA at the P and A sites of the ribosome influences the accuracy of translation.
The lack of detectable variation when the sequence of a protein is analyzed demonstrates that protein synthesis must be extremely accurate. Very few mistakes are apparent in the form of substitutions of one amino acid for another. There are two general stages in protein synthesis at which errors might be made (see Figure 6.8 in 6.3 Special mechanisms control the accuracy of protein synthesis):
  • Charging a tRNA only with its correct amino acid clearly is critical. This is a function of the aminoacyl-tRNA synthetase. Probably the error rate varies with the particular enzyme, but generally mistakes occur in <1/105 aminoacylations.
  • The specificity of codon-anticodon recognition is crucial, but puzzling. Although binding constants vary with the individual codon-anticodon reaction, the specificity is always much too low to provide an error rate of <105. When free in solution, tRNAs bind to their trinucleotide codon sequences only rather weakly. Related, but erroneous, triplets (with two correct bases out of three) are recognized 101102 times as efficiently as the correct triplets.
Codon-anticodon base pairing therefore seems to be a weak point in the accuracy of translation. The ribosome has an important role in controlling the specificity of this interaction, functioning directly or indirectly as a "proofreader," to distinguish correct and incorrect codon-anticodon pairs, and thus amplifying the rather modest intrinsic difference by ~1000×. And in addition to the role of the ribosome itself, the factors that place initiator- and aminoacyl-tRNAs in the ribosome also may influence the pairing reaction.
So there must be some mechanism for stabilizing the correct aminoacyl-tRNA, allowing its amino acid to be accepted as a substrate for receipt of the polypeptide chain; contacts with an incorrect aminoacyl-tRNA must be rapidly broken, so that the complex leaves without reacting. Suppose that there is no specificity in the initial collision between the aminoacyl-tRNA·EF-Tu·GTP complex and the ribosome. If any complex, irrespective of its tRNA, can enter the A site, the number of incorrect entries must far exceed the number of correct entries.
There are two basic models for how the ribosome might discriminate between correctly and incorrectly paired aminoacyl-tRNAs. The actual situation incorporates elements of both models.
  • The direct recognition model supposes that the structure of the ribosome is designed to recognize aminoacyl-tRNAs that are correctly paired. This would mean that the correct pairing results in some small change in the conformation of the aminoacyl-tRNA that the ribosome can recognize. Discrimination occurs before any further reaction occurs.
  • The kinetic proofreading model proposes that there are two (or more) stages in the process, so that the aminoacyl-tRNA has multiple opportunities to disengage. An incorrectly paired aminoacyl-tRNA may pass through some stages of the reaction before it is rejected. Overall selectivity can in principle be the product of the selectivities at each stage.
Figure 7.27 illustrates diagrammatically what happens to correctly and incorrectly paired aminoacyl-tRNAs. A correctly paired aminoacyl-tRNA is able to make stabilizing contacts with rRNA. An incorrectly paired aminoacyl-tRNA does not make these contacts, and therefore is able to diffuse out of the A site.
The path to discovering these interactions started with investigations of the effects of the antibiotic streptomycin in the 1960s. Streptomycin inhibits protein synthesis by binding to 16S rRNA and inhibiting the ability of EF-G to catalyze translocation. It also increases the level of misreading of the pyrimidines U and C (usually one is mistaken for the other, occasionally for A). The site at which streptomycin acts is influenced by the S12 protein; the sequence of this protein is altered in resistant mutants. Ribosomes with an S12 protein derived from resistant bacteria show a reduction in the level of misreading compared with wild-type ribosomes. In effect, S12 controls the level of misreading. When it is mutated to decrease misreading, it suppresses the effect of streptomycin.
S12 stabilizes the structure of 16S rRNA in the region that is bound by streptomycin. The important point to note here is that the P/A site region influences the accuracy of translation: translation can be made more or less accurate by changing the structure of 16S rRNA. The combination of the effects of the S12 protein and streptomycin on the rRNA structure explains the behavior of different mutants in S12, some of which even make the ribosome dependent on the presence of streptomycin for correct translation (Carter et al., 2000).
We now know from the crystal structure of the ribosome that 16S rRNA is in a position to make contacts with aminoacyl-tRNA (for review see Ramakrishnan, 2002). Two bases of 16S rRNA can contact the minor groove of the helix formed by pairing between the anticodon in tRNA with the first two bases of the codon in mRNA (Ogle et al., 2001). This directly stabilizes the structure when the correct codon-anticodon contacts are made at the first two codon positions, but it does not monitor contacts at the third position.
The stabilization of correctly paired aminoacyl-tRNA may have two effects. By holding the aminoacyl-tRNA in the A site, it prevents it from escaping before the next stage of protein synthesis. And the conformational change in the rRNA may help to trigger the next stage of the reaction, which is the hydrolysis of GTP by EF-Tu.
Part of the proofreading effect is determined by timing. An aminoacyl-tRNA in the A site may in effect be trapped if the next stage of protein synthesis occurs while it is there. So a delay between entry into the A site and peptidyl transfer may give more opportunity for a mismatched aminoacyl-tRNA to dissociate. Mismatched aminoacyl-tRNA dissociates more rapidly than correctly matched aminoacyl-tRNA, probably by a factor of ~5×. Its chance of escaping is therefore increased when the peptide transfer step is slowed (for review see Kurland, 1992).
The specificity of decoding has been assumed to reside with the ribosome itself, but some recent results suggest that translation factors influence the process at both the P site and A site. An indication that EF-Tu is involved in maintaining the reading frame is provided by mutants of the factor that suppress frameshifting. This implies that EF-Tu does not merely bring aminoacyl-tRNA to the A site, but also is involved in positioning the incoming aminoacyl-tRNA relative to the peptidyl-tRNA in the P site.
A striking case where factors influence meaning is found at initiation. Mutation of the AUG initiation codon to UUG in the yeast gene HIS4 prevents initiation. Extragenic suppressor mutations can be found that allow protein synthesis to be initiated at the mutant UUG codon. Two of these suppressors prove to be in genes coding for the α and β subunits of eIF2, the factor that binds Met-tRNAi to the P site. The mutation in eIFβ2 resides in a part of the protein that is almost certainly involved in binding nucleic acid. It seems likely that its target is either the initiation sequence of mRNA as such or the base-paired association between the mRNA codon and tRNAiMet anticodon. This suggests that eIF2 participates in the discrimination of initiation codons as well as bringing the initiator tRNA to the P site.
The cost of protein synthesis in terms of high-energy bonds may be increased by proofreading processes. An important question in calculating the cost of protein synthesis is the stage at which the decision is taken on whether to accept a tRNA. If a decision occurs immediately to release an aminoacyl-tRNA·EF-Tu·GTP complex, there is little extra cost for rejecting the large number of incorrect tRNAs that are likely (statistically) to enter the A site before the correct tRNA is recognized. But if GTP is hydrolyzed before the mismatched aminoacyl-tRNA dissociates, the cost will be greater. A mismatched aminoacyl-tRNA can be rejected either before or after the cleavage of GTP, although we do not know yet where on average it is rejected. There is some evidence that the use of GTP in vivo is greater than the three high-energy bonds that are used in adding every (correct) amino acid to the chain


Suppressors may compete with wild-type reading of the code


KEY TERMS:
  • The context of a codon in mRNA refers to the fact that neighboring sequences may change the efficiency with which a codon is recognized by its aminoacyl-tRNA or is used to terminate protein synthesis.
  • Readthrough at transcription or translation occurs when RNA polymerase or the ribosome, respectively, ignores a termination signal because of a mutation of the template or the behavior of an accessory factor.
KEY CONCEPTS:
  • Suppressor tRNAs compete with wild-type tRNAs that have the same anticodon to read the corresponding codon(s).
  • Efficient suppression is deleterious because it results in readthrough past normal termination codons.
  • The UGA codon is leaky and is misread by Trp-tRNA at 1-3% frequency. 

There is an interesting difference between the usual recognition of a codon by its proper aminoacyl-tRNA and the situation in which mutation allows a suppressor tRNA to recognize a new codon. In the wild-type cell, only one meaning can be attributed to a given codon, which represents either a particular amino acid or a signal for termination. But in a cell carrying a suppressor mutation, the mutant codon has the alternatives of being recognized by the suppressor tRNA or of being read with its usual meaning.
A nonsense suppressor tRNA must compete with the release factors that recognize the termination codon(s). A missense suppressor tRNA must compete with the tRNAs that respond properly to its new codon. The extent of competition influences the efficiency of suppression; so the effectiveness of a particular suppressor depends not only on the affinity between its anticodon and the target codon, but also on its concentration in the cell, and on the parameters governing the competing termination or insertion reactions.
The efficiency with which any particular codon is read is influenced by its location. So the extent of nonsense suppression by a given tRNA can vary quite widely, depending on the context of the codon. We do not understand the effect that neighboring bases in mRNA have on codon-anticodon recognition, but the context can change the frequency with which a codon is recognized by a particular tRNA by more than an order of magnitude. The base on the 3 side of a codon appears to have a particularly strong effect.

A nonsense suppressor is isolated by its ability to respond to a mutant nonsense codon. But the same triplet sequence constitutes one of the normal termination signals of the cell! The mutant tRNA that suppresses the nonsense mutation must in principle be able to suppress natural termination at the end of any gene that uses this codon. Figure 7.26 shows that this readthrough results in the synthesis of a longer protein, with additional C-terminal material. The extended protein will end at the next termination triplet sequence found in the phase of the reading frame. Any extensive suppression of termination is likely to be deleterious to the cell by producing extended proteins whose functions are thereby altered.
Amber suppressors tend to be relatively efficient, usually in the range of 10-50%, depending on the system. This efficiency is possible because amber codons are used relatively infrequently to terminate protein synthesis in E. coli.
Ochre suppressors are difficult to isolate. They are always much less efficient, usually with activities below 10%. All ochre suppressors grow rather poorly, which indicates that suppression of both UAA and UAG is damaging to E. coli, probably because the ochre codon is used most frequently as a natural termination signal.
UGA is the least efficient of the termination codons in its natural function; it is misread by Trp-tRNA as frequently as 1-3% in wild-type situations. In spite of this deficiency, however, it is used more commonly than the amber triplet to terminate bacterial genes.
One gene's missense suppressor is likely to be another gene's mutator. A suppressor corrects a mutation by substituting one amino acid for another at the mutant site. But in other locations, the same substitution will replace the wild-type amino acid with a new amino acid. The change may inhibit normal protein function.
This poses a dilemma for the cell: it must suppress what is a mutant codon at one location, while failing to change too extensively its normal meaning at other locations. The absence of any strong missense suppressors is therefore explained by the damaging effects that would be caused by a general and efficient substitution of amino acids.
A mutation that creates a suppressor tRNA can have two consequences. First, it allows the tRNA to recognize a new codon. Second, sometimes it prevents the tRNA from recognizing the codons to which it previously responded. It is significant that all the high-efficiency amber suppressors are derived by mutation of one copy of a redundant tRNA set. In these cases, the cell has several tRNAs able to respond to the codon originally recognized by the wild-type tRNA. So the mutation does not abolish recognition of the old codons, which continue to be served adequately by the tRNAs of the set. In the unusual situation in which there is only a single tRNA that responds to a particular codon, any mutation that prevents the response is lethal (for review see Murgola, 1985; Eggertsson and Soll, 1988; Normanly and Abelson, 1989; Atkins, 1991).
Suppression is most often considered in the context of a mutation that changes the reading of a codon. However, there are some situations in which a stop codon is read as an amino acid at a low frequency in the wild-type situation. The first example to be discovered was the coat protein gene of the RNA phage Qβ. The formation of infective Qβ particles requires that the stop codon at the end of this gene is suppressed at a low frequency to generate a small proportion of coat proteins with a C-terminal extension. In effect, this stop codon is leaky. The reason is that Trp-tRNA recognizes the codon at a low frequency (Hirsh, 1971; Weiner and Weber, 1973).
Readthrough past stop codons occurs also in eukaryotes, where it is employed most often by RNA viruses. This may involve the suppression of UAG/UAA by Tyr-tRNA, Gln-tRNA, or Leu-tRNA, or the suppression of UGA by Trp-tRNA or Arg-tRNA. The extent of partial suppression is dictated by the context surrounding the codon (for review see Beier and Grimm, 2001).

There are nonsense suppressors for each termination codon


KEY CONCEPTS:
  • Each type of nonsense codon is suppressed by tRNAs with mutant anticodons.
  • Some rare suppressor tRNAs have mutations in other parts of the molecule. 
Nonsense suppressors fall into three classes, one for each type of termination codon. Figure 7.25 describes the properties of some of the best characterized suppressors.
The easiest to characterize have been amber suppressors. In E. coli, at least 6 tRNAs have been mutated to recognize UAG codons. All of the amber suppressor tRNAs have the anticodon CUA, in each case derived from wild type by a single base change. The site of mutation can be any one of the three bases of the anticodon, as seen from supD, supE, and supF. Each suppressor tRNA recognizes only the UAG codon, instead of its former codon(s). The amino acids inserted are serine, glutamine, or tyrosine, the same as those carried by the corresponding wild-type tRNAs.
Ochre suppressors also arise by mutations in the anticodon. The best known are supC and supG, which insert tyrosine or lysine in response to both ochre (UAA) and amber (UAG) codons. This conforms with the prediction of the wobble hypothesis that UAA cannot be recognized alone.
A UGA suppressor has an unexpected property. It is derived from tRNATrp, but its only mutation is the substitution of A in place of G at position 24. This change replaces a G·U pair in the D stem with an A·U pair, increasing the stability of the helix. The sequence of the anticodon remains the same as the wild type, CCA . So the mutation in the D stem must in some way alter the conformation of the anticodon loop, allowing CCA to pair with UGA in an unusual wobble pairing of C with A. The suppressor tRNA continues to recognize its usual codon, UGG.
A related response is seen with a eukaryotic tRNA. Bovine liver contains a tRNASer with the anticodon mCCA. The wobble rules predict that this tRNA should respond to the tryptophan codon UGG; but in fact it responds to the termination codon UGA. So it is possible that UGA is suppressed naturally in this situation.
The general importance of these observations lies in the demonstration that codon-anticodon recognition of either wild-type or mutant tRNA cannot be predicted entirely from the relevant triplet sequences, but is influenced by other features of the molecule.

Suppressor tRNAs have mutated anticodons that read new codons


KEY TERMS:
  • A suppressor is a second mutation that compensates for or alters the effects of a primary mutation.
  • A nonsense suppressor is a gene coding for a mutant tRNA able to respond to one or more of the termination codons and insert an amino acid at that site.
  • A missense suppressor codes for a tRNA that has been mutated so as to recognize a different codon. By inserting a different amino acid at a mutant codon, the tRNA suppresses the effect of the original mutation.
KEY CONCEPTS:
  • A suppressor tRNA typically has a mutation in the anticodon that changes the codons to which it responds.
  • When the new anticodon corresponds to a termination codon, an amino acid is inserted and the polypeptide chain is extended beyond the termination codon. This results in nonsense suppression at a site of nonsense mutation or in readthrough at a natural termination codon.
  • Missense suppression occurs when the tRNA recognizes a different codon from usual, so that one amino acid is substituted for another.  

Isolation of mutant tRNAs has been one of the most potent tools for analyzing the ability of a tRNA to respond to its codon(s) in mRNA, and for determining the effects that different parts of the tRNA molecule have on codon-anticodon recognition.
Mutant tRNAs are isolated by virtue of their ability to overcome the effects of mutations in genes coding for proteins. In general genetic terminology, a mutation that is able to overcome the effects of another mutation is called a suppressor.
In tRNA suppressor systems, the primary mutation changes a codon in an mRNA so that the protein product is no longer functional. The secondary, suppressor mutation changes the anticodon of a tRNA, so that it recognizes the mutant codon instead of (or as well as) its original target codon. The amino acid that is now inserted restores protein function. The suppressors are described as nonsense suppressors or missense suppressors, depending on the nature of the original mutation.

In a wild-type cell, a nonsense mutation is recognized only by a release factor, terminating protein synthesis. The suppressor mutation creates an aminoacyl-tRNA that can recognize the termination codon; by inserting an amino acid, it allows protein synthesis to continue beyond the site of nonsense mutation. This new capacity of the translation system allows a full-length protein to be synthesized, as illustrated in Figure 7.23. If the amino acid inserted by suppression is different from the amino acid that was originally present at this site in the wild-type protein, the activity of the protein may be altered.
Missense mutations change a codon representing one amino acid into a codon representing another amino acid, one that cannot function in the protein in place of the original residue. (Formally, any substitution of amino acids constitutes a missense mutation, but in practice it is detected only if it changes the activity of the protein.) The mutation can be suppressed by the insertion either of the original amino acid or of some other amino acid that is acceptable to the protein.













Figure 7.24 demonstrates that missense suppression can be accomplished in the same way as nonsense suppression, by mutating the anticodon of a tRNA carrying an acceptable amino acid so that it responds to the mutant codon. So missense suppression involves a change in the meaning of the codon from one amino acid to another.

Synthetases use proofreading to improve accuracy


KEY TERMS:
  • Proofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chain.
  • Kinetic proofreading describes a proofreading mechanism that depends on incorrect events proceeding more slowly than correct events, so that incorrect events are reversed before a subunit is added to a polymeric chain.
  • Chemical proofreading describes a proofreading mechanism in which the correction event occurs after addition of an incorrect subunit to a polymeric chain, by reversing the addition reaction.
KEY CONCEPTS:
  • Specificity of recognition of both amino acid and tRNA is controlled by aminoacyl-tRNA synthetases by proofreading reactions that reverse the catalytic reaction if the wrong component has been incorporated.  

Aminoacyl-tRNA synthetases have a difficult job. Each synthetase must distinguish 1 out of 20 amino acids, and and must differentiate cognate tRNAs (typically 1-3) from the total set (perhaps 100 in all).
Many amino acids are closely related to one another, and all amino acids are related to the metabolic intermediates in their particular synthetic pathway. It is especially difficult to distinguish between two amino acids that differ only in the length of the carbon backbone (that is, by one CH2 group). Intrinsic discrimination based on relative energies of binding two such amino acids would be only ~1/5. The synthetase enzymes improve this ratio ~1000 fold.
Intrinsic discrimination between tRNAs is better, because the tRNA offers a larger surface with which to make more contacts, but it is still true that all tRNAs conform to the same general structure, and there may be a quite limited set of features that distinguish the cognate tRNAs from the noncognate tRNAs.
We can imagine two general ways in which the enzyme might select its substrate:
  • The cycle of admittance, scrutiny, rejection/acceptance could represent a single binding step that precedes all other stages of whatever reaction is involved. This is tantamount to saying that the affinity of the binding site is sufficient to control the entry of substrate. In the case of synthetases, this would mean that only the correct amino acids and cognate tRNAs could form a stable attachment at the site.
  • Alternatively, the reaction proceeds through some of its stages, after which a decision is reached on whether the correct species is present. If it is not present, the reaction is reversed, or a bypass route is taken, and the wrong member is expelled. This sort of postbinding scrutiny is generally described as proofreading. In the example of synthetases, it would require that the charging reaction proceeds through certain stages even if the wrong tRNA or amino acid is present.
Synthetases use proofreading mechanisms to control the recognition of both types of substrates. They improve significantly on the intrinsic differences among amino acids or among tRNAs, but, consistent with the intrinsic differences in each group, make more mistakes in selecting amino acids (error rates are 104 - 105) than in selecting tRNAs (error rates are ~10-6) (see Figure 6.8).

Transfer RNA binds to synthetase by the two stage reaction depicted in Figure 7.18. Cognate tRNAs have a greater intrinsic affinity for the binding site, so they are bound more rapidly and dissociate more slowly. Following binding, the enzyme scrutinizes the tRNA that has been bound. If the correct tRNA is present, binding is stabilized by a conformational change in the enzyme. This allows aminoacylation to occur rapidly. If the wrong tRNA is present, the conformational change does not occur. As a result, the reaction proceeds much more slowly; this increases the chance that the tRNA will dissociate from the enzyme before it is charged. This type of control is called kinetic proofreading (Hopfield, 1974).
Specificity for amino acids varies among the synthetases. Some are highly specific for initially binding a single amino acid, but others can also activate amino acids closely related to the proper substrate. Although the analog amino acid can sometimes be converted to the adenylate form, in none of these cases is an incorrectly activated amino acid actually used to form a stable aminoacyl-tRNA.
The presence of the cognate tRNA usually is needed to trigger proofreading, even if the reaction occurs at the stage before formation of aminoacyl-adenylate. (An exception is provided by Met-tRNA synthetase, which can reject noncognate aminoacyl-adenylate complexes even in the absence of tRNA.) 









There are two stages at which proofreading of an incorrect aminoacyl-adenylate may occur during formation of aminoacyl-tRNA. Figure 7.19 shows that both use chemical proofreading, in which the catalytic reaction is reversed. The extent to which one pathway or the other predominates varies with the individual synthetase:
  • The noncognate aminoacyl-adenylate may be hydrolyzed when the cognate tRNA binds. This mechanism is used predominantly by several synthetases, including those for methionine, isoleucine, and valine. (Usually, the reaction cannot be seen in vivo, but it can be followed for Met-tRNA synthetase when the incorrectly activated amino acid is homocysteine, which lacks the methyl group of methionine). Proofreading releases the amino acid in an altered form, as homocysteine thiolactone. In fact, homocysteine thiolactone is produced in E. coli as a by-product of the charging reaction of Met-tRNA synthetase. This shows that continuous proofreading is part of the process of charging a tRNA with its amino acid (Jakubowski, 1990).
  • Some synthetases use chemical proofreading at a later stage. The wrong amino acid is actually transferred to tRNA, is then recognized as incorrect by its structure in the tRNA binding site, and so is hydrolyzed and released. The process requires a continual cycle of linkage and hydrolysis until the correct amino acid is transferred to the tRNA. 
A classic example in which discrimination between amino acids depends on the presence of tRNA is provided by the Ile-tRNA synthetase of E. coli. The enzyme can charge valine with AMP, but hydrolyzes the valyl-adenylate when tRNAIle is added. The overall error rate depends on the specificities of the individual steps, as summarized in Figure 7.20. The overall error rate of 1.5 × 105 is less than the measured rate at which valine is substituted for isoleucine (in rabbit globin), which is 2-5 × 104. So mischarging probably provides only a small fraction of the errors that actually occur in protein synthesis.

Ile-tRNA synthetase uses size as a basis for discrimination among amino acids. Figure 7.21 shows that it has two active sites: the synthetic (or activation) site and the editing (or hydrolytic) site. The crystal structure of the enzyme shows that the synthetic site is too small to allow leucine (a close analog of isoleucine) to enter. All amino acids large than isoleucine are excluded from activation because they cannot enter the synthetic site. An amino acid that can enter the synthetic site is placed on tRNA. Then the enzyme tries to transfer it to the editing site. Isoleucine is safe from editing because it is too large to enter the editing site. However, valine can enter this site, and as a result an incorrect Val-tRNAIle is hydrolyzed. Essentially the enzyme provides a double molecular sieve, in which size of the amino acid is used to discriminate between closely related species (Nureki et al., 1998; for review see Jakubowski and Goldman, 1992).



One interesting feature of Ile-tRNA synthetase is that the synthetic and editing sites are a considerable distance apart, ~34Å. A crystal structure of the enzyme complexed with an edited analog of isoleucine shows that the amino acid is transported from the synthetic site to the editing site (Silvian, Wang, and Steitz, 1999). Figure 7.22 shows that this involves a change in the conformation of the tRNA. The amino acid acceptor stem of tRNAIle can exist in alternative conformations. It adopts an unusual hairpin in order to be aminoacylated by an amino acid in the synthetic site. Then it returns to the more common helical structure in order to move the amino acid to the editing site. The translocation between sites is the rate-limiting step in proofreading (Nomanbhoy, Hendrickson, and Schimmel, 1999). Ile-tRNA synthetase is a class I synthetase, but the double sieve mechanism is used also by class II synthetases (Dock-Bregeon et al., 2000).

Aminoacyl-tRNA synthetases fall into two groups


KEY CONCEPTS:
  • Aminoacyl-tRNA synthetases are divided into the class I and class II groups by sequence and structural similarities.
In spite of their common function, synthetases are a rather diverse group of proteins. The individual subunits vary from 40-110 kD, and the enzymes may be monomeric, dimeric, or tetrameric. Homologies between them are rare. Of course, the active site that recognizes tRNA comprises a rather small part of the molecule. It is interesting to compare the active sites of different synthetases.

Synthetases have been divided into two general groups, each containing 10 enzymes, on the basis of the structure of the domain that contains the active site. A general type of organization that applies to both groups is represented in Figure 7.14. The catalytic domain includes the binding sites for ATP and amino acid. It can be recognized as a large region that is interrupted by an insertion of the domain that binds the acceptor helix of the tRNA. This places the terminus of the tRNA in proximity to the catalytic site. A separate domain binds the anticodon region of tRNA. Those synthetases that are multimeric also possess an oligomerization domain (for review see Schimmel, 1987).
Class I synthetases have an N-terminal catalytic domain that is identified by the presence of two short, partly conserved sequences of amino acids, sometimes called "signature sequences." The catalytic domain takes the form of a motif called a nucleotide-binding fold (which is also found in other classes of enzymes that bind nucleotides). The nucleotide fold consists of alternating parallel β-strands and α-helices; the signature sequence forms part of the ATP-binding site. The insertion that contacts the acceptor helix of tRNA differs widely between different class I enzymes. The C-terminal domains of the class I synthetases, which include the tRNA anticodon-binding domain and any oligomerization domain, also are quite different from one another.
Class II enzymes share three rather general similarities of sequence in their catalytic domains. The active site contains a large antiparallel β-sheet surrounded by α-helices. Again, the acceptor helix-binding domain that interrupts the catalytic domain has a structure that depends on the individual enzyme. The anticodon-binding domain tends to be N-terminal. The location of any oligomerization domain is widely variable.
The lack of any apparent relationship between the two groups of synthetases is a puzzle. Perhaps they evolved independently of one another. This makes it seem possible even that an early form of life could have existed with proteins that were made up of just the 10 amino acids coded by one type or the other. 

A general model for synthetase·tRNA binding suggests that the protein binds the tRNA along the "side" of the L-shaped molecule. The same general principle applies for all synthetase·tRNA binding: the tRNA is bound principally at its two extremities, and most of the tRNA sequence is not involved in recognition by a synthetase. However, the detailed nature of the interaction is different between class I and class II enzymes, as can be seen from the models of Figure 7.15, which are based on crystal structures. The two types of enzyme approach the tRNA from opposite sides, with the result that the tRNA-protein models look almost like mirror images of one another.

A class I enzyme (Gln-tRNA synthetase) approaches the D-loop side of the tRNA. It recognizes the minor groove of the acceptor stem at one end of the binding site, and interacts with the anticodon loop at the other end. Figure 7.16 is a diagrammatic representation of the crystal structure of the tRNAGln·synthetase complex. A revealing feature of the structure is that contacts with the enzyme change the structure of the tRNA at two important points. These can be seen by comparing the dotted and solid lines in the anticodon loop and acceptor stem:
  • Bases U35 and U36 in the anticodon loop are pulled farther out of the tRNA into the protein.
  • The end of the acceptor stem is seriously distorted, with the result that base pairing between U1 and A72 is disrupted. The single-stranded end of the stem pokes into a deep pocket in the synthetase protein, which also contains the binding site for ATP.
This structure explains why changes in U35, G73, or the U1-A72 base pair affect the recognition of the tRNA by its synthetase. At all of these positions, hydrogen bonding occurs between the protein and tRNA (Rould et al., 1989).

A class II enzyme (Asp-tRNA synthetase) approaches the tRNA from the other side, and recognizes the variable loop, and the major groove of the acceptor stem, as drawn in Figure 7.17. The acceptor stem remains in its regular helical conformation. ATP is probably bound near to the terminal adenine. At the other end of the binding site, there is a tight contact with the anticodon loop, which has a change in conformation that allows the anticodon to be in close contact with the protein (Ruff et al., 1991).


tRNAs are charged with amino acids by synthetases


KEY TERMS:
  • Cognate tRNAs (Isoaccepting tRNA) are those recognized by a particular aminoacyl-tRNA synthetase. They all are charged with the same amino acid.
KEY CONCEPTS:
  • Aminoacyl-tRNA synthetases are enzymes that charge tRNA with an amino acid to generate aminoacyl-tRNA in a two-stage reaction that uses energy from ATP.
  • There are 20 aminoacyl-tRNA synthetases in each cell. Each charges all the tRNAs that represent a particular amino acid.
  • Recognition of a tRNA is based on a small number of points of contact in the tRNA sequence. 

It is necessary for tRNAs to have certain characteristics in common, yet be distinguished by others. The crucial feature that confers this capacity is the ability of tRNA to fold into a specific tertiary structure. Changes in the details of this structure, such as the angle of the two arms of the "L" or the protrusion of individual bases, may distinguish the individual tRNAs.
All tRNAs can fit in the P and A sites of the ribosome, where at one end they are associated with mRNA via codon-anticodon pairing, while at the other end the polypeptide is being transferred. Similarly, all tRNAs (except the initiator) share the ability to be recognized by the translation factors (EF-Tu or eEF1) for binding to the ribosome. The initiator tRNA is recognized instead by IF-2 or eIF2. So the tRNA set must possess common features for interaction with elongation factors, but the initiator tRNA can be distinguished.

Amino acids enter the protein synthesis pathway through the aminoacyl-tRNA synthetases, which provide the interface for connection with nucleic acid. All synthetases function by the two-step mechanism depicted in Figure 7.13:
  • First, the amino acid reacts with ATP to form aminoacyl~adenylate, releasing pyrophosphate. Energy for the reaction is provided by cleaving the high energy bond of the ATP.
  • Then the activated amino acid is transferred to the tRNA, releasing AMP.
The synthetases sort the tRNAs and amino acids into corresponding sets. Each synthetase recognizes a single amino acid and all the tRNAs that should be charged with it. Usually, each amino acid is represented by more than one tRNA. Several tRNAs may be needed to respond to synonym codons, and sometimes there are multiple species of tRNA reacting with the same codon. Multiple tRNAs representing the same amino acid are called isoaccepting tRNAs; because they are all recognized by the same synthetase, they are also described as its cognate tRNAs.
Many attempts to deduce similarities in sequence between cognate tRNAs, or to induce chemical alterations that affect their charging, have shown that the basis for recognition is different for different tRNAs, and does not necessarily lie in some feature of primary or secondary structure alone. We know from the crystal structure that the acceptor stem and the anticodon stem make tight contacts with the synthetase, and mutations that alter recognition of a tRNA are found in these two regions. (The anticodon itself is not necessarily recognized as such; for example, the "suppressor" mutations discussed later in this chapter change a base in the anticodon, and therefore the codons to which a tRNA responds, without altering its charging with amino acids.)
A group of isoaccepting tRNAs must be charged only by the single aminoacyl-tRNA synthetase specific for their amino acid. So isoaccepting tRNAs must share some common feature(s) enabling the enzyme to distinguish them from the other tRNAs. The entire complement of tRNAs is divided into 20 isoaccepting groups; each group is able to identify itself to its particular synthetase.
tRNAs are identified by their synthetases by contacts that recognize a small number of bases, typically from 1-5. Three types of feature commonly are used:
  • Usually (but not always), at least one base of the anticodon is recognized. Sometimes all the positions of the anticodon are important.
  • Often one of the last three base pairs in the acceptor stem is recognized. An extreme case is represented by alanine tRNA, which is identified by a single unique base pair in the acceptor stem.
  • The so-called discriminator base, which lies between the acceptor stem and the CCA terminus, is always invariant among isoacceptor tRNAs.
No one of these features constitutes a unique means of distinguishing 20 sets of tRNAs, or provides sufficient specificity, so it appears that recognition of tRNAs is idiosyncratic, each following its own rules.
Several synthetases can specifically charge a "minihelix" consisting only of the acceptor and Tψ C arms (equivalent to one arm of the L-shaped molecule) with the correct amino acid. For certain tRNAs, specificity depends exclusively upon the acceptor stem. However, it is clear that there are significant variations between tRNAs, and in some cases the anticodon region is important. Mutations in the anticodon can affect recognition by the class II Phe-tRNA synthetase. Multiple features may be involved; minihelices from the tRNAVal and tRNAMet (where we know that the anticodon is important in vivo) can react specifically with their synthetases.
So recognition depends on an interaction between a few points of contact in the tRNA, concentrated at the extremities, and a few amino acids constituting the active site in the protein. The relative importance of the roles played by the acceptor stem and anticodon is different for each tRNA·synthetase interaction (for review see Schimmel, 1989).