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 tRNA^Gly 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 tRNA^Lys 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 reason—most likely steric hindrance—the 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 × 10^–5 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 sort—such as a nonsense codon or frameshift—to 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:

• Suppression involves recognition of a codon by a (mutant) tRNA that usually would respond to a different codon (see 7.12 Suppressor tRNAs have mutated anticodons that read new codons).
• Redefinition of the meaning of a codon occurs when an aminoacyl-tRNA is modified (see 7.8 Novel amino acids can be inserted at certain stop codons).

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 <10^–5. 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 10^–1–10^–2 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-tRNA_i 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 tRNA_i^Met 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).