Functions of DEAD-box proteins in bacteria: current knowledge and pending questions.

DEAD-box proteins are RNA-dependent ATPases that are widespread in all three kingdoms of life. They are thought to rearrange the structures of RNA or ribonucleoprotein complexes but their exact mechanism of action is rarely known. Whereas in yeast most DEAD-box proteins are essential, no example of an essential bacterial DEAD-box protein has been reported so far; at most, their absence results in cold-sensitive growth. Moreover, whereas yeast DEAD-box proteins are implicated in virtually all reactions involving RNA, in E. coli (the bacterium where DEAD-box proteins have been mostly studied) their role is limited to ribosome biogenesis, mRNA degradation, and possibly translation initiation. Plausible reasons for these differences are discussed here. In spite of their dispensability, E. coli DEAD-box proteins are valuable models for the mechanism of action of DEAD-box proteins in general because the reactions in which they participate can be reproduced in vitro. Here we review our present understanding of this mechanism of action. Using selected examples for which information is available: (i) we describe how, by interacting directly with a particular RNA motif or by binding to proteins that themselves recognize such a motif, DEAD-box proteins are brought to their specific RNA substrate(s); (ii) we discuss the nature of the structural transitions that DEAD-box proteins induce on their substrates; and (iii) we analyze the reasons why these proteins are mostly important at low temperatures. This article is part of a Special Issue entitled: The Biology of RNA helicases-Modulation for life.

Probing ribosomal protein-RNA interactions with an external force.

Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites--i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations--i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by "one-side" clamping.

Termination troubles in Escherichia coli K12.

In this issue of Molecular Microbiology, Schaub and Hayes report that, compared with other enterobacteria, Escherichia coli K12 carries two mutations - one in the prfB gene encoding the release factor RF2, and the other in the rpsG gene encoding r-protein S7 - that together concur in compromising translation termination at the essential rpsG gene. As a consequence, the growth of E. coli K12 is very sensitive to a further mutation (rluD(-) ) that depresses RF2 activity, whereas the growth of its close relative, E. coli B, is not. We tentatively discuss how the K12-specific mutations in RF2 and S7 might have occurred and why inefficient translation termination at rpsG inhibits growth. The work of Schaub and Hayes illustrates the fact that, due probably to its long history in the laboratory, E. coli K12 has accumulated mutations that sometimes limit its value as a model for studying basic steps in prokaryotic gene expression.

Identification of the sites of action of SrmB, a DEAD-box RNA helicase involved in Escherichia coli ribosome assembly.

DEAD-box RNA-dependent ATPases are ubiquitous enzymes that participate in nearly all processes involving RNA, but their detailed molecular functions remain generally unknown. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit notably by facilitating the incorporation of L13, one of the ribosomal proteins that bind 23S rRNA earliest. Previously, we showed that SrmB is tethered to nascent ribosome through interactions with L4, L24 and the region from domain I of 23S rRNA that binds them. To identify the sites of action of SrmB, we have characterized rRNA mutations that bypass SrmB requirement. Five of them affect the same position from two repeated heptanucleotides in domain II of 23S rRNA, whereas two others affect a complementary hexanucleotide in 5S rRNA. Thus the sites of action of SrmB differ from its tethering site. In the mature ribosome, one of the heptanucleotides participates in a highly compact structure that contacts L13, the '1024 G-ribo wrench'. In addition, we have observed that the assembly defect of ΔsrmB cells worsens as rRNA synthesis increases. Based on these results, we propose two non-exclusive scenarios for the role of SrmB in ribosome assembly.

SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo.

DEAD-box proteins play specific roles in remodeling RNA or ribonucleoprotein complexes. Yet, in vitro, they generally behave as nonspecific RNA-dependent ATPases, raising the question of what determines their specificity in vivo. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit. Moreover, when overexpressed, it compensates for a mutation in L24, the ribosomal protein (r-protein) thought to initiate assembly. Here, using the tandem affinity purification (TAP) procedure, we show that SrmB forms a complex with r-proteins L4, L24 and a region near the 5'-end of 23S rRNA that binds these proteins. In vitro reconstitution experiments show that the stability of this complex reflects cooperative interactions of SrmB with L4, L24 and rRNA. These observations are consistent with an early role of SrmB in assembly and explain the genetic link between SrmB and L24. Besides its catalytic core, SrmB possesses a nonconserved C-terminal extension that, we show, is not essential for SrmB function and specificity. In this regard, SrmB differs from DbpA, another DEAD-box protein involved in ribosome assembly.

Cold adaptation in DEAD-box proteins.

Spontaneous rearrangements of RNA structures are usually characterized by large activation energies and thus become very slow at low temperatures, yet RNA structure must remain dynamic even in cold-adapted (psychrophilic) organisms. DEAD-box proteins constitute a ubiquitous family of RNA-dependent ATPases that can often unwind short RNA duplexes in vitro (helicase activity), hence the belief that one of their major (though not exclusive) roles in vivo is to assist in RNA rearrangements. Here, we compare two Escherichia coli DEAD-box proteins and their orthologs from the psychrophilic bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea from the point of view of enzymatic properties. One of these proteins (SrmB) is involved in ribosome assembly, whereas the other (RhlE) presumably participates in both mRNA degradation and ribosome assembly; in vitro, RhlE is far more active as a helicase than SrmB. The activation energy associated with the ATPase activity of the psychrophilic SrmB is lower than for its mesophilic counterpart, making it more active at low temperatures. In contrast, in the case of psychrophilic RhlE, it is the RNA unwinding activity, not the ATPase activity, that has a reduced activation energy and is therefore cold-adapted. We argue that these different modes of cold adaptation reflect the likely function of these proteins in vivo: RNA helicase for RhlE and ATP-dependent RNA binding for SrmB. The cold adaptation of helicases like RhlE presumably facilitates RNA metabolism in psychrophilic bacteria.

DEAD-box RNA helicases in Escherichia coli.

In spite of their importance in RNA metabolism, the function of DExD/H-box proteins (including DEAD-box proteins) is poorly understood at the molecular level. Here, we present recent progress achieved with the five DEAD-box proteins from Escherichia coli, which have been particularly well studied. These proteins, which have orthologues in many bacteria, participate, in particular, in specific steps of mRNA decay and ribosome assembly. In vitro, they behave as poorly processive RNA helicases, presumably because they only unwind a few base pairs at each cycle so that stable duplexes can reanneal rather than dissociate. Except for one of them (DbpA), these proteins lack RNA specificity in vitro, and specificity in vivo is likely conferred by partners that target them to defined substrates. Interestingly, at least one of them is multifunctional, presumably because it can interact with different partners. Altogether, several aspects of the information gathered with these proteins have become paradigms for our understanding of DEAD-box proteins in general.

Troubleshooting coupled in vitro transcription-translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins.

Cell-free coupled transcription-translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 +/- 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20 degrees C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20 degrees C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.

CsdA, a cold-shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit.

CsdA, a DEAD-box protein from Escherichia coli, has been proposed to participate in a variety of processes, such as translation initiation, gene regulation after cold-shock, mRNA decay and biogenesis of the small ribosomal subunit. Whether the protein really plays a direct role in these multiple processes is however, not clear. Here, we show that CsdA is involved in the biogenesis of the large rather than the small ribosomal subunit. Deletion of the csdA gene leads to a deficit in free 50S subunits at low temperatures and to the accumulation of a new particle sedimenting around 40S. Analysis of the RNA and protein contents of this particle indicates that it corresponds to a mis-assembled large subunit. Sucrose gradient fractionation shows that in wild-type cells CsdA associates mainly with a pre50S particle. Presumably the RNA helicase activity of CsdA permits a structural rearrangement during 50S biogenesis at low temperature. We showed previously that SrmB, another DEAD-box RNA helicase, is also involved in 50S assembly in E.coli. Our results suggest that CsdA is required at a later step than SrmB. However, over-expression of CsdA corrects the ribosome defect of the srmB-deleted strain, indicating that some functional overlap exists between the two proteins.

A FRET-based, continuous assay for the helicase activity of DEAD-box proteins.

Enzymes from cold-adapted organisms are generally endowed with lower activation enthalpies than their counterparts from organisms growing at higher temperatures, making them better catalysts in the cold. However, the enzymes of RNA metabolism have not been examined in this respect. A challenge for studying cold adaptation of DEAD-box RNA helicases is the low precision of the classical, discontinuous helicase assay based on electrophoretic separation of duplexes and isolated strands. Here, we describe a continuous, FRET-based assay that allows the measurement of the helicase activities of DEAD-box proteins with a precision high enough to detect changes in activation enthalpies associated with cold adaptation.

Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP.

Killer and protective ribosomes.

In prokaryotes, translation influences mRNA decay. The breakdown of most Escherichia coli mRNAs is initiated by RNase E, a 5'-dependent endonuclease. Some mRNAs are protected by ribosomes even if these are located far upstream of cleavage sites ("protection at a distance"), whereas others require direct shielding of these sites. I argue that these situations reflect different modes of interaction of RNase E with mRNAs. Protection at a distance is most impressive in Bacilli, where ribosomes can protect kilobases of unstable downstream sequences. I propose that this protection reflects the role in mRNA decay of RNase J1, a 5'-->3' exonuclease with no E. coli equivalent. Finally, recent years have shown that besides their protective role, ribosomes can also cleave their mRNA under circumstances that cause ribosome stalling. The endonuclease associated with this "killing" activity, which has a eukaryotic counterpart ("no-go decay"), is not characterized; it may be borne by the distressed ribosome itself.

Construction and characterization of E. coli K12 strains in which the transcription of selected genes is desynchronized from translation.

In Escherichia coli, synthesis and translation of individual mRNAs are usually synchronous, so that no long ribosome-free mRNA stretch exists between the RNA polymerase and the leading ribosome. By comparing situations in which the same mRNA (the lacZ mRNA) is synthesized either by the genuine E. coli RNA polymerase or the faster T7 RNA polymerase, we have previously shown that the outpacing of ribosomes by RNA polymerase destabilizes mRNAs, and more so as outpacing becomes larger. This destabilization requires the noncatalytic C-terminal region of RNase E; more generally, there is circumstantial evidence that this region is specifically involved in the fast decay of various untranslated mRNAs. The genetic system designed for desynchronizing transcription and translation with T7 RNA polymerase was originally designed in the E. coli B strain BL21(DE3). Here, we describe procedures for transferring this system to the more common E. coli K12 background. We also show that it can be used as a screen for identifying factors involved in the instability of untranslated mRNA. Protocols in use in this laboratory for RNA extraction, Northern blotting, and beta-galactosidase assay are described and critically discussed.

The deleterious effect of an insertion sequence removing the last twenty percent of the essential Escherichia coli rpsA gene is due to mRNA destabilization, not protein truncation.

Ribosomal protein S1, the product of the essential rpsA gene, consists of six imperfect repeats of the same motif. Besides playing a critical role in translation initiation on most mRNAs, S1 also specifically autoregulates the translation of its own messenger. ssyF29 is a viable rpsA allele that carries an IS10R insertion within the coding sequence, resulting in a protein lacking the last motif (S1DeltaC). The growth of ssyF29 cells is slower than that of wild-type cells. Moreover, translation of a reporter rpsA-lacZ fusion is specifically stimulated, suggesting that the last motif is necessary for autoregulation. However, in ssyF29 cells the rpsA mRNA is also strongly destabilized; this destabilization, by causing S1DeltaC shortage, might also explain the observed slow-growth and autoregulation defect. To fix this ambiguity, we have introduced an early stop codon in the rpsA chromosomal gene, resulting in the synthesis of the S1DeltaC protein without an IS10R insertion (rpsADeltaC allele). rpsADeltaC cells grow much faster than their ssyF29 counterparts; moreover, in these cells S1 autoregulation and mRNA stability are normal. In vitro, the S1DeltaC protein binds mRNAs (including its own) almost as avidly as wild-type S1. These results demonstrate that the last S1 motif is dispensable for translation and autoregulation: the defects seen with ssyF29 cells reflect an IS10R-mediated destabilization of the rpsA mRNA, probably due to facilitated exonucleolytic degradation.

The highly efficient translation initiation region from the Escherichia coli rpsA gene lacks a shine-dalgarno element.

The translational initiation region (TIR) of the Escherichia coli rpsA gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position -91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the "specialized ribosome" approach. Depending upon the constructs used, translation initiation was decreased three- to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins.

A mutation in T7 RNA polymerase that facilitates promoter clearance.

Like multisubunit RNA polymerases (RNAPs), T7 RNAP frequently releases its transcript over the initial 8-12 transcribed nucleotides, when it still contacts the promoter. This abortive cycling, which is most prominent with initial sequences that deviate from those of T7 late genes, eventually compromises productive transcription. Starting from an in vivo situation where transcription of a target gene by T7 RNAP is virtually abolished because of extensive abortive cycling, we have selected a mutation in RNAP that restores target gene expression. In vitro, this mutation (P266L) weakens promoter binding but markedly reduces abortive cycling over a variety of initial sequences by stabilizing the transcription complex at nucleotides 5-8. Other substitutions of P266 have similar effects. X-ray data show that during the transition from initial to elongation complex, the N-terminal region undergoes a major structural switch of which P266 constitutes one of the hinges. How the mutation might facilitate this switch is tentatively discussed. On the practical side, the mutation can significantly improve in vitro transcription, particularly from templates carrying unfavorable initial sequences.

AU-rich sequences within 5' untranslated leaders enhance translation and stabilize mRNA in Escherichia coli.

We have shown previously that when the Escherichia coli chromosomal lacZ gene is put under the control of an extended Shine-Dalgarno (SD) sequence (10 or 6 nucleotides in length), the translation efficiency can be highly variable, depending on the presence of AU-rich targets for ribosomal protein S1 in the mRNA leader. Here, the same strains have been used to examine the question of how strong ribosome binding to extended SD sequences affects the stability of lacZ mRNAs translated with different efficiencies. The steady-state concentration of the lacZ transcripts has been found to vary over a broad range, directly correlating with translation efficiency but not with the SD duplex stability. The observed strain-to-strain variations in lacZ mRNA level became far less marked in the presence of the rne-1 mutation, which partially inactivates RNase E. Together, the results show that (i) an SD sequence, even one that is very long, cannot stabilize the lacZ mRNA in E. coli if translation is inefficient; (ii) inefficiently translated lacZ transcripts are sensitive to RNase E; and (iii) AU-rich elements inserted upstream of a long SD sequence enhance translation and stabilize mRNA, despite the fact that they constitute potential RNase E sites. These data strongly support the idea that the lacZ mRNA in E. coli can be stabilized only by translating, and not by stalling, ribosomes.

The poly(A) tail of mRNAs: bodyguard in eukaryotes, scavenger in bacteria.

In eukaryotes, poly(A) tails usually act as stabilizers of intact mRNAs, whereas in E. coli they serve to accelerate the destruction of fragments. The mechanisms underlying these contrasting effects of the same RNA modification are discussed.

Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases.

DEAD-box proteins participate in various aspects of RNA metabolism in all organisms. These RNA-dependent ATPases are usually regarded as double-stranded RNA unwinding enzymes, though in vitro this activity has only been demonstrated for a subset of them. Given their high biological specificity, their equivocal unwinding activity may reflect the noncognate character of the substrates used in vitro. Here, we pinpoint other reasons for this elusiveness. We have compared the ATPase and helicase activities of three E. coli DEAD-box proteins, CsdA, RhlE and SrmB. Whereas the ATPase activity of all proteins is stimulated (albeit to various degree) by long RNAs, only RhlE is stimulated by short oligoribonucleotides. Consistently, all three proteins can unwind RNA duplexes with long single-stranded extensions, but only RhlE is effective when extensions are short or absent. Another critical constraint concerns the length of the duplex region: in the case of RhlE, the ratio (duplex unwound)/(ATP hydrolyzed) drops 1000-fold upon going from 11 to 14 base pairs, indicating a low processivity. Remarkably, allowing for these constraints, all three proteins can unwind substrates with either 5' or 3' extensions (or no extension in the case of RhlE). This behavior, which contrasts with that of well studied SF1 DNA helicases, is discussed in the light of available structural and biochemical data.

The histone-like protein HU does not obstruct movement of T7 RNA polymerase in Escherichia coli cells but stimulates its activity.

In vivo, RNA polymerases (RNAPs) do not transcribe naked DNA but do transcribe protein-associated DNA. Studies with the model enzyme T7 RNAP have shown that, in eukaryotic cells or in vitro, nucleosomes can inhibit both transcription initiation and elongation. We examine here whether the presence of HU, one of the major histone-like proteins in Escherichia coli cells (the genuine milieu for T7 RNAP) affects its activity. An engineered lac operon fused to the T7 late promoter was introduced into the chromosome of T7 RNAP-producing strains that either overexpress HU or lack it. The flows of RNAP that enter and exit this operon were compared with regard to the content of HU. We found that the fraction of T7 RNAP molecules that do not reach the end of the lac operon (ca. 15%) is the same whether the host cells overexpressed HU or lacked it: thus, the enzyme either freely displaces HU or transcribes through it. However, in these cells, the transcript yield was increased when HU is overexpressed and decreased in the hup mutants, presumably reflecting changes in DNA supercoiling. Thus, in contrast to eukaryotic nucleosomes, HU does not impair T7 RNAP activity but has a stimulatory effect. Finally, our results suggest that HU can also influence mRNA stability in vivo.