Simultaneous and functional binding of SmpB and EF-Tu-TP to the alanyl acceptor arm of tmRNA.

Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by specific cellular proteases. For Escherichia coli, previous studies on initial steps of this "trans-translation" mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for subsequent delivery to stalled ribosomes are inefficient when compared to analogous reactions with canonical tRNA(Ala). In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans-translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu-GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the acceptor arm of Ala-tRNA(Ala), but there the alanylation efficiency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation.

Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis.

The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets proteins for degradation by ClpXP and ClpAP. Mutational analysis of the ssrA tag reveals independent, but overlapping determinants for its interactions with ClpX, ClpA, and SspB, a specificity-enhancing factor for ClpX. ClpX interacts with residues 9-11 at the C terminus of the tag, whereas ClpA recognizes positions 8-10 in addition to residues 1-2 at the N terminus. SspB interacts with residues 1-4 and 7, N-terminal to the ClpX-binding determinants, but overlapping the ClpA determinants. As a result, SspB and ClpX work together to recognize ssrA-tagged substrates efficiently, whereas SspB inhibits recognition of these substrates by ClpA. Thus, dissection of the recognition signals within the ssrA tag provides insight into how multiple proteins function in concert to modulate proteolysis.

Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine.

ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.

Identification of endogenous SsrA-tagged proteins reveals tagging at positions corresponding to stop codons.

The SsrA.SmpB quality control system adds a C-terminal degradation peptide (AANDENYALAA) to nascent chains on stalled ribosomes, thereby freeing the ribosome and ensuring proteolysis of the tagged protein. An SsrA mutant with the tag sequence AANDEHHHHHH was used to slow degradation and facilitate Ni2+-nitrilotriacetic acid affinity purification. Display of affinity-purified Escherichia coli proteins on two-dimensional gels revealed small quantities of a diverse set of SsrA-H6-tagged proteins, and mass spectroscopy identified LacI repressor, lambda cI repressor, YbeL, GalE, RbsK, and a SlyD-kan(R) fusion protein as members of this set. For lambda repressor and YbeL, the SsrA-H6 tag was added after the natural C terminus of the protein, suggesting that tagging occurred while the ribosome idled at the termination codon of these genes. Potential causes of tagging for the other proteins include interference from translation of downstream reading frames, rare codons, and gene disruption. These and previous results support a broad role for the SsrA.SmpB system in freeing stalled ribosomes and in directing degradation of the products of these frustrated protein synthesis reactions.

Characterization of the N-terminal repeat domain of Escherichia coli ClpA-A class I Clp/HSP100 ATPase.

The ClpA, ClpB, and ClpC subfamilies of the Clp/HSP100 ATPases contain a conserved N-terminal region of approximately 150 residues that consists of two approximate sequence repeats. This sequence from the Escherichia coli ClpA enzyme is shown to encode an independent structural domain (the R domain) that is monomeric and approximately 40% alpha-helical. A ClpA fragment lacking the R domain showed ATP-dependent oligomerization, protein-stimulated ATPase activity, and the ability to complex with the ClpP peptidase and mediate degradation of peptide and protein substrates, including casein and ssrA-tagged proteins. Compared with the activities of the wild-type ClpA, however, those of the ClpA fragment missing the R domain were reduced. These results indicate that the R domain is not required for the basic recognition, unfolding, and translocation functions that allow ClpA-ClpP to degrade some protein substrates, but they suggest that it may play a role in modulating these activities.

Protein factors associated with the SsrA.SmpB tagging and ribosome rescue complex.

SsrA RNA acts as a tRNA and mRNA to modify proteins whose synthesis on ribosomes has stalled. Such proteins are marked for degradation by addition of peptide tags to their C termini in a reaction mediated by SsrA RNA and SmpB, a specific SsrA-RNA binding protein. Evidence is presented here for the existence of a larger ribonucleoprotein complex that contains ribosomal protein S1, phosphoribosyl pyrophosphate synthase, RNase R, and YfbG in addition to SsrA RNA and SmpB. Biochemical, genetic, and phylogenetic results suggest potential roles for some of these factors in various stages of the ribosome rescue and tagging process and/or the presence of functional interactions between one or more of these proteins and SsrA.

Altering dimerization specificity by changes in surface electrostatics.

Arc repressor forms a homodimer in which the subunits intertwine to create a single globular domain. To obtain Arc sequences that fold preferentially as heterodimers, variants with surface patches of excess positive or negative charge were designed. Several but not all oppositely charged sequence pairs showed preferential heterodimer formation. In the most successful design pair, alpha helix B of one subunit contained glutamic acids at positions 43, 46, 47, 48, and 50, whereas the other subunit contained lysines or arginines at these positions. A continuum electrostatic model captures many features of the experimental results and suggests that the most successful designs include elements of both positive and negative design.

Molecular determinants of complex formation between Clp/Hsp100 ATPases and the ClpP peptidase.

The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals. Many family members also interact with peptidases to form ATP-dependent proteases. In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase. Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition. Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities. Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner. Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.

Contributions of distinct quaternary contacts to cooperative operator binding by Mnt repressor.

Mnt, a tetrameric repressor encoded by bacteriophage P22, uses N-domain dimers to contact each half of its operator site. Experiments with a double mutant and structural homology with the P22 Arc repressor suggest that contacts made by Arg-28 and stabilized by Glu-33 are largely responsible for dimer-dimer cooperativity in Mnt. These dimer-dimer contacts are energetically more important for operator binding than solution tetramerization, which is mediated by an independent C-terminal coiled-coil domain. Indeed, once one dimer of the Mnt tetramer contacts an operator half site, binding of the second dimer occurs with an effective concentration much lower than that expected if both dimers were flexibly tethered. These results suggest that binding of the second dimer introduces some strain into the protein-DNA complex, a mechanism that could serve to limit the affinity of operator binding and to prevent strong binding of the Mnt tetramer to nonoperator sites.

An evolutionary bridge to a new protein fold.

Arc repressor bearing the N11L substitution (Arc-N11L) is an evolutionary intermediate between the wild type protein, in which the region surrounding position 11 forms a beta-sheet, and a double mutant 'switch Arc', in which this region is helical. Here, Arc-N11L is shown to be able to adopt either the wild type or mutant conformations. Exchange between these structures occurs on the millisecond time scale in a dynamic equilibrium in which the relative populations of each fold depend on temperature, solvent conditions and ligand binding. The N11L mutation serves as an evolutionary bridge from the beta-sheet to the helical fold because in the mutant, Leu is an integral part of the hydrophobic core of the new structure but can also occupy a surface position in the wild type structure. Conversely, the polar Asn 11 side chain serves as a negative design element in wild type Arc because it cannot be incorporated into the core of the mutant fold.

Striking stabilization of Arc repressor by an engineered disulfide bond.

A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the antiparallel strands of the beta sheet both in the Arc repressor dimer and in a single-chain variant in which the Arc subunits are connected by a 15-residue peptide tether. In both proteins, the presence of the disulfide bond increased the T(m) by approximately 40 degrees C. In the single-chain background, the disulfide bond stabilized Arc by 8.5 kcal/mol relative to the reduced form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabilization arises from a modest effective concentration of the Cys11-Cys11' disulfide in the native state (71 M) and an anomalously low effective concentration in the denatured state (40 microM). Disulfide cross-linking of the two beta strands in the single-chain Arc background accelerated refolding by a factor of 170 into the sub-microsecond time scale. However, the major energetic effect of the disulfide occurs after the transition state for Arc refolding, slowing unfolding by 200 000-fold.

Interactions of Arg2 in the Mnt N-terminal arm with the central and flanking regions of the Mnt operator.

Arg2, in the N-terminal arm of the Mnt repressor, plays an important role in determining operator-binding specificity. In the complex of the Mnt tetramer with the 21 base-pair mnt operator, there are four potential sites for Arg2 interactions, two in the central region of the operator and two on the outer flanks of the operator. Single-chain variants of the dimeric N-terminal domain of Mnt containing one Arg2 residue and one Lys2 or Met2 residue were constructed and interactions with operator DNA were probed using Fe. EDTA affinity cleavage. The results of these orientation studies show that the majority of the energetically significant interactions mediated by Arg2 occur in the central region of the mnt operator. The RK2, RA2, and RM2 mutations reduce the free energy of operator binding by 1.7 kcal/mol, 3.3 kcal/mol, and 4.9 kcal/mol, respectively. Double-mutant thermodynamic cycle analyses using the RA2, RM2, and operator variants also reveal interaction free energies between Arg2 and operator base-pairs 9, 10, 11, 12 and 13, which in aggregate account for most of the Arg2 contribution to operator binding.

A specificity-enhancing factor for the ClpXP degradation machine.

Events that stall bacterial protein synthesis activate the ssrA-tagging machinery, resulting in resumption of translation and addition of an 11-residue peptide to the carboxyl terminus of the nascent chain. This ssrA-encoded peptide tag marks the incomplete protein for degradation by the energy-dependent ClpXP protease. Here, a ribosome-associated protein, SspB, was found to bind specifically to ssrA-tagged proteins and to enhance recognition of these proteins by ClpXP. Cells with an sspB mutation are defective in degrading ssrA-tagged proteins, demonstrating that SspB is a specificity-enhancing factor for ClpXP that controls substrate choice.

Dynamics of substrate denaturation and translocation by the ClpXP degradation machine.

ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.

Evidence for partial secondary structure formation in the transition state for arc repressor refolding and dimerization.

Structure formation and dimerization are concerted processes in the refolding of Arc repressor. The integrity of secondary structure in the transition state of Arc refolding has been investigated here by determining the changes in equilibrium stability and refolding/unfolding kinetics for a set of Ala --> Gly mutations at residues that are solvent-exposed in the native Arc dimer. At some sites, reduced stability was caused primarily by faster unfolding, indicating that secondary structure at these positions is largely absent in the transition state. However, most of the Ala --> Gly substitutions in the alpha-helices of Arc and a triple mutant in the beta-sheet also resulted in decreased refolding rates, in some cases, accounting for the major fraction of thermodynamic destabilization. Overall, these results suggest that some regions of native secondary structure are present but incompletely formed in the transition state of Arc refolding and dimerization. Consolidation of this secondary structure, like close packing of the hydrophobic core, seems to occur later in the folding process. On average, Phi(F) values for the Ala --> Gly mutations were significantly larger than Phi(F) values previously determined for alanine-substitution mutants, suggesting that backbone interactions in the transition state may be stronger than side chain interactions. Mutations causing significant reductions in the Arc refolding rate were found to cluster in the central turn of alpha-helix A and in the first two turns of alpha-helix B. In the Arc dimer, these elements pack together in a compact structure, which might serve as nucleus for further folding.

The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue.

Bacteria contain a remarkable RNA molecule - known alternatively as SsrA RNA, tmRNA, or 10Sa RNA - that acts both as a tRNA and as an mRNA to direct the modification of proteins whose biosynthesis has stalled or has been interrupted. These incomplete proteins are marked for degradation by cotranslational addition of peptide tags to their C-termini in a reaction that is mediated by ribosome-bound SsrA RNA and an associated protein factor, SmpB. This system plays a key role in intracellular protein quality control and also provides a mechanism to clear jammed or obstructed ribosomes. Here the structural, functional and phylogenetic properties of this unique RNA and its associated factors are reviewed, and the intracellular proteases that act to degrade the proteins tagged by this system are also discussed.

Regulation of high affinity nickel uptake in bacteria. Ni2+-Dependent interaction of NikR with wild-type and mutant operator sites.

Escherichia coli actively imports nickel via the ATP-dependent NikABCDE permease. NikR, a protein of the ribbon-helix-helix family of transcription factors, represses expression of the nikABCDE operon in the presence of excessive concentrations of intracellular nickel. Here, the NikR operator site is identified within the nikABCDE promoter by footprinting and mutational analyses. The operator consists of two dyad-symmetric 5'-GTATGA-3' recognition sequences separated by 16 base pairs. Mutations in the GTATGA sequences reduce NikR binding affinity in vitro and reduce repression of a P(nik)-lacZ fusion in vivo. Moreover, NikR is shown to be a direct sensor of nickel ions. Strong operator binding requires the continual presence of 20-50 micrometer nickel, indicating the presence of a low affinity nickel-binding site, and NikR dimers also contain two high affinity nickel-binding sites. In addition to both GTATGA sites and nickel, high affinity operator binding also requires the C-terminal domain of NikR.

Structure and dynamics of the tetrameric mnt repressor and a model for its DNA complex.

Abstract The tetrameric Mnt repressor of bacteriophage P22 consists of two dimeric DNA-binding domains and a tetramerization domain. The NOE and chemical shift data demonstrate that the structures of the domains in the wild-type repressor protein are similar to those of the separate domains, the three-dimensional structures of which have been determined previously. (15)N relaxation measurements show that the linker that connects the anti-parallel four-helix bundle with the two ß-sheet DNA-binding dimers is highly flexible. No evidence was found for interactions between the distinct modules. The (15)N relaxation properties of the two domains differ substantially, confirming their structural independence. A model in which one two-stranded coiled coil of the four-helix bundle is attached to one N-terminal dimer is most consistent with the biochemical data and (15)N relaxation data. For the Mnt-DNA complex this geometry fits with a model in which the two ß-sheet DNA-binding domains are bound at two successive major grooves of the Mnt operator and the tetramerization domain is packed between these two DNA-bound dimers. In such a model the two-fold symmetry axis of the four-helix bundle coincides with that of the operator sequence and the two bound dimers. Bending of the Mnt operator of approximately 30° upon binding of the tetramer, as measured by gel-shift assays, is in agreement with this model of the Mnt-DNA complex.