Autogenous translation regulation by Escherichia coli ATPase SecA may be mediated by an intrinsic RNA helicase activity of this protein.

The seven conserved motifs typical of the helicase superfamily II have been identified in the sequences of Escherichia coli protein SecA, an ATPase mediating protein translocation across the inner membrane of the bacterium, and its Bacillus subtilis homolog Div. It is hypothesized that SecA and Div possess an RNA helicase activity and may couple ATP hydrolysis both to membrane translocation of proteins, and to hairpin unwinding in their own mRNAs, leading to the known autogenous regulation of translation.     

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase

The SecA ATPase is a protein translocase motor and a superfamily 2 (SF2) RNA helicase. The ATPase catalytic core ('DEAD motor') contains the seven conserved SF2 motifs. Here, we demonstrate that Motif III is essential for SecA-mediated protein translocation and viability. SecA Motif III mutants can bind ligands (nucleotide, the SecYEG translocase 'channel', signal and mature preprotein domains), can catalyse basal and SecYEG-stimulated ATP hydrolysis and can be activated for catalysis. However, Motif III mutation specifically blocks the preprotein-stimulated 'translocation ATPase' at a step of the reaction pathway that lies downstream of ligand binding. A functional Motif III is required for optimal ligand-driven conformational changes and kinetic parameters that underlie optimal preprotein-modulated nucleotide cycling at the SecA DEAD motor. We propose that helicase Motif III couples preprotein binding to the SecA translocation ATPase and that catalytic activation of SF2 enzymes through Motif-III-mediated action is essential for both polypeptide and nucleic-acid substrates.     

Control of SecA and SecM translation by protein secretion

SecA, the protein translocation ATPase of E. coli is subject to secretion-defect-response control. SecM (secretion monitor) encoded by the 5' region of the secM-secA mRNA is involved in this regulation. SecM translation is subject to transient elongation arrest at Pro166, which is prolonged when export of the nascent SecM is blocked. An "arrest sequence", FXXXXWIXXXXGIRAGP, was identified at a carboxy-terminal region of SecM that interacts with the ribosomal exit tunnel. Presumably, the stalled ribosome disrupts the secondary structure of the secM-secA mRNA such that the Shine-Dalgarno sequence for translation of secA is exposed. Mutation studies established that the SecM elongation arrest is required for the viability of E. coli as well as for constitutive (in secretion-proficient cells) and upregulated (in secretion compromised cells) expression of SecA. Furthermore, evidence suggests that elongation-arresting SecM has a role of upregulating the functionality of newly synthesized SecA molecules, presumably by bringing the mRNA to the vicinity of the membrane/Sec translocation apparatus. These results are discussed in relation to the versatile nature of SecA in its localization and structure.     

SecA: the ubiquitous component of preprotein translocase in prokaryotes

SecA is an obligatory component of the complex hetero-septameric translocase of prokaryotes. It is unique in that it exists as two forms within the holoenzyme; first, as a structural component of the preprotein channel and second, as an ATP-dependent membrane cycling factor facilitating the translocation of a broad class of proteins across the cytoplasmic membrane. While the translocase activity of SecA appears to be functionally conserved, it is not clear whether the mechanisms of regulation of the secA gene are similarly maintained. The recent characterization of an ATP-dependent RNA helicase activity of SecA offers a unique mechanism for SecA to communicate the secretion status of the cell to the appropriate regulatory circuits simply by the unwinding of an appropriate RNA target. Resolution of these two activities through combined biochemical, genetic, and biophysical studies should lead to a better understanding of the role of SecA in bacterial secretion.     

Isolation and analysis of dominant secA mutations in Escherichia coli.

The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.     

SecA protein: Autoregulated initiator of secretory precursor protein translocation across theE. coli plasma membrane

Several classes ofsecA mutants have been isolated which reveal the essential role of this gene product forE. coli cell envelope protein secretion. SecA-dependent,in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulatesecA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.     

Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function

SecA, the motor subunit of bacterial polypeptide translocase, is an RNA helicase. SecA comprises a dimerization C-terminal domain fused to an ATPase N-terminal domain containing conserved DEAD helicase motifs. We show that the N-terminal domain is organized like the motor core of DEAD proteins, encompassing two subdomains, NBD1 and IRA2. NBD1, a rigid nucleotide-binding domain, contains the minimal ATPase catalytic machinery. IRA2 binds to NBD1 and acts as an intramolecular regulator of ATP hydrolysis by controlling ADP release and optimal ATP catalysis at NBD1. IRA2 is flexible and can undergo changes in its alpha-helical content. The C-terminal domain associates with NBD1 and IRA2 and restricts IRA2 activator function. Thus, cytoplasmic SecA is maintained in the thermally stabilized ADP-bound state and unnecessary ATP hydrolysis cycles are prevented. Two DEAD family motifs in IRA2 are essential for IRA2-NBD1 binding, optimal nucleotide turnover and polypeptide translocation. We propose that translocation ligands alleviate C-terminal domain suppression, allowing IRA2 to stimulate nucleotide turnover at NBD1. DEAD motors may employ similar mechanisms to translocate different enzymes along chemically unrelated biopolymers.     

Characterization of Escherichia coli SecA protein binding to a site on its mRNA involved in autoregulation.

In order to understand further the autogenous regulation of Escherichia coli secA translation, we have set up a purified system to study the binding of SecA protein to portions of its mRNA. Specific SecA protein-RNA binding was demonstrated by UV cross-linking, filter binding, and gel shift assays. Use of the filter binding assay allowed optimization of binding, which was influenced by Mg2+ and ATP concentrations, and a measurement of the affinity of this interaction. A nested series of RNAs lacking either 5' or 3' portions of geneX-secA sequences were used to localize the SecA protein binding site to sequences around the geneX-secA intergenic region. These studies imply that SecA protein directly regulates its own translation by a specific RNA binding activity that presumably blocks translational initiation.     

Secretion monitor, SecM, undergoes self-translation arrest in the cytosol

The product of the Escherichia coli secM gene (secretion monitor, formerly gene X), upstream of secA, is involved in secretion-responsive control of SecA translation. In wild-type cells, SecM is rapidly degraded by the periplasmic tail-specific protease. It is also subject to a transient translation pause at a position close to the C terminus. The elongation arrest was strikingly prolonged when translocation of SecM was impaired. SRP was not required for this arrest. Instead, the nascent SecM product itself may participate, as the arrest was diminished when it incorporated a proline analog, azetidine. We propose that cytosolically localized nascent SecM undergoes self-translation arrest, thereby enhancing translation of secA through an altered secondary structure of the secM-secA messenger RNA.     

Competition between ribosome and SecA binding promotes Escherichia coli secA translational regulation.

SecA protein, the protein translocation ATPase of Escherichia coli, autogenously regulates its translation during normal protein secretion by binding to a secretion-responsive element located near the 5' end of its gene on geneX-secA mRNA. In order to characterize this autoregulation further, RNA footprinting and primerextension inhibition (toeprinting) studies were carried out with a segment of geneX-secA RNA, 30S ribosomal subunits and tRNAfMet along with purified SecA protein. The results show that ribosome and SecA-binding sites overlap, indicating that a simple competition for binding of geneX-secA mRNA presumably governs the translation initiation step. Further analysis showed that SecA protein was able to specifically dissociate a preformed 30S-tRNAfMet-geneX-secA RNA ternary complex as indicated by the disappearance of its characteristic toeprint after SecA addition. These findings are consistent with secA autoregulation, and they suggest a novel mechanism for the autoregulatory behavior of this complex protein.     

Role of p54 RNA Helicase Activity and Its C-terminal Domain in Translational Repression, P-body Localization and Assembly

The RNA helicase p54 (DDX6, Dhh1, Me31B, Cgh-1, RCK) is a prototypic component of P-(rocessing) bodies in cells ranging from yeast to human. Previously, we have shown that it is also a component of the large cytoplasmic polyadenylation element-binding protein translation repressor complex in Xenopus oocytes and that when tethered to the 3′ untranslated region, Xp54 represses reporter mRNA translation. Here, we examine the role of the p54 helicase activity in translational repression and in P-body formation. Mutagenesis of conserved p54 helicase motifs activates translation in the tethered function assay, reduces accumulation of p54 in P-bodies in HeLa cells, and inhibits its capacity to assemble P-bodies in p54-depleted cells. Similar results were obtained in four helicase motifs implicated in ATP binding and in coupling ATPase and RNA binding activities. This is accompanied by changes in the interaction of the mutant p54 with the oocyte repressor complex components. Surprisingly, the C-terminal D2 domain alone is sufficient for translational repression and complete accumulation in P-bodies, although it is deficient for P-body assembly. We propose a novel RNA helicase model, in which the D2 domain acts as a protein binding platform and the ATPase/helicase activity allows protein complex remodeling that dictates the balance between repressors and an activator of translation.     

Critical regions of secM that control its translation and secretion and promote secretion-specific secA regulation

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3' end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.     

In vivo membrane topology of Escherichia coli SecA ATPase reveals extensive periplasmic exposure of multiple functionally important domains clustering on one face of SecA

The Sec-dependent protein translocation pathway promotes the transport of proteins into or across the bacterial plasma membrane. SecA ATPase has been shown to be a nanomotor that associates with its protein cargo as well as the SecYEG channel complex and to undergo ATP-driven cycles of membrane insertion and retraction that promote stepwise protein translocation. Previous studies have shown that both the 65-kDa N-domain and 30-kDa C-domain of SecA appear to undergo such membrane cycling. In the present study we performed in vivo sulfhydryl labeling of an extensive collection of monocysteine secA mutants under topologically specific conditions to identify regions of SecA that are accessible to the trans side of the membrane in its membrane-integrated state. Our results show that distinct regions of five of six SecA domains were labeled under these conditions, and such labeling clusters to a single face of the SecA structure. Our results demarcate an extensive face of SecA that interacts with SecYEG and is in fluid contact with the protein-conducting channel. The observed domain-specific labeling patterns should also provide important constraints on model building efforts in this dynamic system.     

Clostridium difficile has two parallel and essential Sec secretion systems

Protein translocation across the cytoplasmic membrane is an essential process in all bacteria. The Sec system, comprising at its core an ATPase, SecA, and a membrane channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second parallel Sec system has been described in a number of gram-positive species. This accessory Sec system is characterized by the presence of a second copy of the energizing ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a subset of Sec substrates. In common with many pathogenic gram-positive species, Clostridium difficile possesses two copies of SecA. Here, we describe the first characterization of the C. difficile accessory Sec system and the identification of its major substrates. Using inducible antisense RNA expression and dominant-negative alleles of secA1 and secA2, we demonstrate that export of the S-layer proteins (SLPs) and an additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic precursor of the SLPs SlpA and other cell wall proteins was observed in cells expressing dominant-negative secA1 or secA2 alleles, concomitant with a decrease in the levels of mature SLPs in the cell wall. Furthermore, expression of either dominant-negative allele or antisense RNA knockdown of SecA1 or SecA2 dramatically impaired growth, indicating that both Sec systems are essential in C. difficile.     

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

The mutation T477A in HIV-1 reverse transcriptase (RT) restores normal proteolytic processing of RT in virus with Gag-Pol mutated in the p51-RNH cleavage site

Background

The zinc-finger antiviral protein (ZAP) specifically inhibits the replication of certain viruses, including murine leukemia virus (MLV), by preventing the accumulation of viral mRNA in the cytoplasm. ZAP directly binds to the viral mRNA through the zinc-finger motifs and recruits the RNA exosome to degrade the target RNA. RNA helicase p72 is required for the optimal function of ZAP. In an attempt to understand the structure-function relationship of ZAP, we performed alanine scanning analysis.

Results

A series of ZAP mutants was generated, in which three consecutive amino acids were replaced with three alanines. The mutants were analyzed for their antiviral activities against pseudotyped MLV vector. Out of the nineteen mutants analyzed, seven displayed significantly lower antiviral activities. Two mutations were in the very N-terminal domain, and five mutations were within or around the first and second zinc-finger motifs. These mutants were further analyzed for their abilities to bind to the target RNA, the exosome, and the RNA helicase p72. Mutants Nm3 and Nm63 lost the ability to bind to RNA. Mutants Nm 63 and Nm93 displayed compromised interaction with p72, while the binding of Nm133 to p72 was very modest. The interactions of all the mutants with the exosome were comparable to wild type ZAP.

Conclusions

The integrity of the very N-terminal domain and the first and second zinc-finger motifs appear to be required for ZAP's antiviral activity. Analyses of the mutants for their abilities to interact with the target RNA and RNA helicase p72 confirmed our previous results. The mutants that bind normally to the target RNA, the exosome, and the RNA helicase p72 may be useful tools for further understanding the mechanism underlying ZAP's antiviral activity.     

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.     

Two Nonredundant SecA Homologues Function in Mycobacteria

The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of Mycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export--the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in Mycobacterium smegmatis, we demonstrate that secA1 is an essential gene. In contrast, secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature of secA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a Delta secA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.     

Identification of the preprotein binding domain of SecA

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.