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The recognition of a tRNA by its aminoacyl-tRNA synthetase is a classic example of the specificity often encountered in biology. Each of the 20 aminoacyl-tRNA synthetases in a cell must distinguish its own set of isoacceptor tRNAs from the many noncognate tRNAs and efficiently catalyze the covalent attachment of the correct amino acid to the 3' end of only these species. Ultimately, the fate of the cell rests on this interaction, as there are no subsequent proof-reading steps in protein synthesis whereby the amino acid is matched against the anticodon to ensure that the proper amino acid is inserted in response to a given codon. How an aminoacyl-tRNA synthetase is able to select its tRNA substrates from a pool of noncognate species sharing similar tertiary structure has been the focus of over 20 years of research. Recent technical refinements in the types of assays used to study this interaction have contributed a wealth of new information to this field, allowing the identification of nucleotides conferring a particular amino acid acceptor identity for a number of tRNAs. The goal of this chapter is to summarize these more recent developments in tRNA recognition.

ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5′-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5′-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.

Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression ofGCN4mRNA. The phenotypes ofgcd10mutants were suppressed by high-copy-numberIMTgenes, encoding initiator methionyl tRNA (tRNAiMet), orLHP1, encoding the yeast homolog of the human La autoantigen. Thegcd10-504mutation led to a reduction in steady-state levels of mature tRNAiMet, attributable to increased turnover rather than decreased synthesis of pre-tRNAiMet. Remarkably, the lethality of aGCD10deletion was suppressed by high-copy-numberIMT4, indicating that its role in expression of mature tRNAiMetis the essential function of Gcd10p. Agcd14-2mutant also showed reduced amounts of mature tRNAiMet, but in addition, displayed a defect in pre-tRNAiMetprocessing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAiMetmaturation. The chromatographic behavior of elongator and initiator tRNAMeton a RPC-5 column indicated that both species are altered structurally ingcd10Δcells, and analysis of base modifications revealed that 1-methyladenosine (m1A) is undetectable ingcd10ΔtRNA. Interestingly,gcd10andgcd14mutations had no effect on processing or accumulation of elongator tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

ClpA, a member of the Clp/Hsp100 family of ATPases, is a molecular chaperone and, in combination with a proteolytic component ClpP, participates in ATP-dependent proteolysis. We investigated the role of ClpA in protein degradation by ClpAP by dissociating the reaction into several discrete steps. In the assembly step, ClpA–ClpP–substrate complexes assemble either by ClpA–substrate complexes interacting with ClpP or by ClpA–ClpP complexes interacting with substrate; ClpP in the absence of ClpA is unable to bind substrates. Assembly requires ATP binding but not hydrolysis. We discovered that ClpA translocates substrates from their binding sites on ClpA to ClpP. The translocation step specifically requires ATP; nonhydrolyzable ATP analogs are ineffective. Only proteins that are degraded by ClpAP are translocated. Characterization of the degradation step showed that substrates can be degraded in a single round of ClpA–ClpP–substrate binding followed by ATP hydrolysis. The products generated are indistinguishable from steady-state products. Taken together, our results suggest that ClpA, through its interaction with both the substrate and ClpP, acts as a gatekeeper, actively translocating specific substrates into the proteolytic chamber of ClpP where degradation occurs. As multicomponent ATP-dependent proteases are widespread in nature and share structural similarities, these findings may provide a general mechanism for regulation of substrate import into the proteolytic chamber.

ClpA, a newly discovered ATP-dependent molecular chaperone, remodels bacteriophage P1 RepA dimers into monomers, thereby activating the latent specific DNA binding activity of RepA. We investigated the mechanism of the chaperone activity of ClpA by dissociating the reaction into several steps and determining the role of nucleotide in each step. In the presence of ATP or a nonhydrolyzable ATP analog, the initial step is the self-assembly of ClpA and its association with inactive RepA dimers. ClpA-RepA complexes form rapidly and at 0°C but are relatively unstable. The next step is the conversion of unstable ClpA-RepA complexes into stable complexes in a time- and temperature-dependent reaction. The transition to stable ClpA-RepA complexes requires binding of ATP, but not ATP hydrolysis, because nonhydrolyzable ATP analogs satisfy the nucleotide requirement. The stable complexes contain approximately 1 mol of RepA dimer per mol of ClpA hexamer and are committed to activating RepA. In the last step of the reaction, active RepA is released upon exchange of ATP with the nonhydrolyzable ATP analog and ATP hydrolysis. Importantly, we discovered that one cycle of RepA binding to ClpA followed by ATP-dependent release is sufficient to convert inactive RepA to its active form.

A new concept in molecular biology that has evolved over the past ten years is that proteins fold with assistance from other proteins, collectively referred to as molecular chaperones. All organisms, from bacteria to humans, possess several classes of highly conserved molecular chaperones, defined generally as proteins that bind to non-native conformations of proteins and facilitate correct folding and to native proteins and promote refolding. Extensive work has shown that the function as well as the structure of chaperones are highly conserved and the results obtained from studies of one organism are usually applicable to other organisms (1–3). This review will focus on several ATP-dependent molecular chaperone systems in Escherichia coli, including (i) DnaK, and its co-chaperones, DnaJ and GrpE, (ii) Clp proteins and (iii) GroEL and its co-chaperone, GroES (summarized in Table 1).

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)