Arch Microbiol (2002) 177 : 433–440 DOI 10.1007/s00203-002-0407-5
MINI-REVIEW
Jörg Stülke
Control of transcription termination in bacteria by RNA-binding proteins that modulate RNA structures
Received: 17 October 2001 / Revised: 1 February 2002 / Accepted: 3 February 2002 / Published online: 3 April 2002 © Springer-Verlag 2002
Abstract During the past few years, our knowledge of gene regulation by RNA-binding proteins has greatly increased. RNA-binding proteins are involved in processes such as protection of RNAs from RNase degradation, prevention of ribosome binding to mRNA, control of formation of secondary structures of the mRNA that permit or prevent translation initiation, and termination/antitermination of transcription in response to external signals. Modulation of transcription termination by RNA-binding proteins involves the formation of alternative structures. One of the structures can act as a transcriptional terminator, while adoption of the alternative structure prevents formation of the terminator and does thus result in transcript elongation. Which of the two structures prevails under a given condition depends on two factors: the intrinsic stability of the alternative structures and the stabilization of one of both by an RNA-binding regulatory protein. Binding of a protein to the nascent mRNA may result in transcript elongation, as is the case for cold-shock proteins or in several catabolic operons. The RNA-binding ability of the RNA-binding proteins is modulated by direct interaction with the inducer, by protein-protein interactions with sensor proteins or by protein phosphorylation. In contrast, in the pyrimidine or tryptophan biosynthetic operons of Bacillus subtilis, the transcriptional terminators are stabilized by RNA-binding proteins resulting in the absence of expression of these operons.
Introduction In bacteria, gene expression is commonly regulated at the level of transcription initiation. Due to the low stability of
J. Stülke (✉) Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany e-mail:
[email protected], Tel.: +49-9131-8528818, Fax: +49-9131-8528082
most bacterial mRNAs, this provides an efficient way to control the synthesis of any given protein. Most frequently, transcription initiation is tightly regulated by alternative sigma factors, repressors, or activators. In several operons, however, transcription is constitutively initiated, but elongation can only proceed under specific conditions. Regulated transcription elongation can be brought about by different mechanisms: (1) by proteins that bind to RNA polymerase and allow the elongation beyond termination signals, or (2) by the formation of alternative mRNA structures that result in either transcription termination or elongation. The formation of alternative RNA structures can in turn be modulated by tRNAs that bind to the mRNAs or by mRNA-binding proteins that support the formation of structures effective in termination or elongation (antitermination). The mechanism of transcriptional termination/antitermination controlled by RNA-binding proteins will be the subject of this review. Due to space limitations, only recent work shall be reviewed here. Several earlier reviews have dealt with aspects of this subject (Henkin 2000; Rutberg 1997).
Antitermination by RNA chaperones: cold-shock proteins as antiterminators When bacteria experience a sudden decrease of temperature, they respond by synthesizing a specific set of proteins including a family of small, highly conserved proteins, the so-called cold-shock proteins (CspA family). The CspA protein accounts for about 10% of total protein synthesis in cold-shocked cells of Escherichia coli (Phadtare et al. 1999). The finding that proteins of the CspA family of Bacillus subtilis are able to bind RNA, and even more so that they specifically bind the leader region of cspB and cspC led to the proposal that these proteins act as RNA chaperones. According to this model, their binding to mRNAs prevents the formation of secondary structures that may occur at low temperature and interfere with translation efficiency. Thus, binding of CSPs to the untranslated regions of mRNAs would be a
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prerequisite for efficient translation (Graumann et al. 1997). The ability of CSPs to destabilize secondary structures in RNAs suggests that they might also prevent the formation of transcriptional terminators and thus act as antiterminator proteins. Indeed, this idea has very recently been verified (Bae et al. 2001). One of the E. coli operons induced by cold shock is the octacistronic metY rpsO operon. This operon contains several transcriptional terminators. Tandem terminators are present downstream of the promoter-proximal gene, metY, and additional termination sites are located downstream of the fourth and seventh genes of the operon, infB and rpsO, respectively. While transciption terminates at 37 °C, full-length transcripts are formed after cold shock (Bae et al. 2001).
Antitermination of carbon and nitrogen catabolic genes and operons There are three main types of transcriptional antiterminators controlling carbohydrate utilization in bacteria: One large family, of which the E. coli BglG protein is the prototype, binds conserved RNA structures and is controlled by phosphorylation by components of the phosphotransferase system (PTS). The second group is represented by the B. subtilis GlpP protein which responds to direct binding of a low-molecular-weight effector. In the third group, amidase synthesis in Pseudomonas aeruginosa is controlled by an antiterminator whose activity is modulated by interaction with a ligand-sensitive protein. Nitrogensource regulation at the level of transcriptional antitermination has been described for the Klebsiella pneumoniae nas operon involved in nitrate assimilation.
Antiterminators controlled by reversible phosphorylation A growing family of transcriptional antiterminators in bacteria is controlled by protein phosphorylation in response to the availability of the respective inducer. These proteins are all composed of three domains, an N-terminal RNAbinding domain (RBD) and two reiterated PTS regulation domains (PRDs) (Manival et al. 1997; Stülke et al. 1998). The best-studied representatives of this family are the BglG antiterminator from E. coli and SacY and LicT from B. subtilis (Amster-Choder and Wright 1992; Tortosa et al. 1997; Schnetz et al. 1996). It is interesting to note that antiterminators of the BglG family are commonly found in gram-positive bacteria where they control several catabolic pathways including sucrose, β-glucoside, lactose, and glucose utilization (Stülke et al. 1998). By contrast, only β-glucoside utilization is controlled by these antiterminators in gram-negative bacteria (Rutberg 1997). The BglG and LicT antiterminator proteins that control expression of the E. coli and B. subtilis bgl operons, respectively, are involved in both substrate induction and the more general mechanism of carbon catabolite repression (Krüger et al. 1996; Görke and Rak 1999).
Fig. 1 Modulation of transcription elongation at a catabolic (ptsGHI) operon in Bacillus subtilis. Constitutively initiated transcription is terminated in the absence of the inducer, glucose. The presence of glucose triggers the activity of the antiterminator protein, GlcT (gray circles). GlcT binding to the RAT sequence prevents the formation of the terminator and results in transcript elongation
To be active as antiterminators, BglG and LicT bind to RNA sequences that have been mapped to an inverted repeat overlapping the terminator [subsequently called RAT, RNA antiterminator (Aymerich and Steinmetz 1992)]. RAT forms a secondary structure that, if bound by antiterminator protein, prevents the formation of the terminator and allows expression of the bgl operon (Fig. 1). The activity of BglG and LicT is controlled by antagonistically acting phosphorylation events by PTS components: in the absence of glucose, the HPr protein can phosphorylate a conserved histidyl residue in the C-terminal copy of the duplicated PRDs (PRD-II) and thereby stimulate the activity of the antiterminators. In the presence of glucose, HPr is either dephosphorylated (E. coli) or trapped in an alternative phosphorylation state (B. subtilis). Thus, the antiterminators are inactive under these conditions (Lindner et al. 1999; Görke and Rak 1999; Stülke et al. 1998). The second mode of control depends on the inducer of the system, salicin: If present, salicin is transported and phosphorylated by EIIBgl. In the absence of salicin, phosphorylated EIIBgl accumulates and this
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triggers phosphorylation of PRD-I resulting in inactivation of the antiterminators (Görke and Rak 1999; Le Coq et al. 1995). Thus, for activity BglG and LicT must be phosphorylated at PRD-II and unphosphorylated at PRD-I. An interesting feature of the phosphorylation of PRDs is the nature of the phosphorylation sites. Unlike many other bacterial proteins which are phosphorylated on serine or aspartate residues, the PRDs are phosphorylated on histidines (Amster-Choder and Wright 1997; Lindner et al. 1999). The activity of BglG and LicT is modulated by phosphorylation, but how can phosphorylation control RNA binding and antitermination activity of the protein? Two forms of the proteins can exist depending on the phosphorylation state: an active dimeric form and an inactive monomeric form. The dimeric antiterminator proteins are active in antitermination (Amster-Choder and Wright 1992; van Tilbeurgh et al. 2001). The observation that the phosphorylation sites in the activated form of the PRDs of LicT are buried in the dimer interface and thus inaccessible to phosphorylation partners suggests major structural changes upon phosphorylation (van Tilbeurgh et al. 2001). The regulatory scheme outlined above is valid for all the antitermination systems that are controlled by PRDcontaining antiterminators. However, there is a major exception: Antiterminators such as B. subtilis SacY and GlcT, controlling genes that are expressed under conditions that result in catabolite repression, are themselves not subject to catabolite control by HPr-dependent phosphorylation. However, multiple phosphorylation of SacY by HPr was observed. Since mutations of the phosphorylation sites in PRD-II of SacY did not affect antitermination, phosphorylation of this domain may occur but be without physiological relavance (Bachem and Stülke 1998; Tortosa et al. 1997). The structural determinants of protein-RNA interactions by antiterminators of the BglG family have been studied most intensively with the RNA-binding domain of SacY from B. subtilis and its cognate sacB RAT sequence. Stem-loop pairing of the sacB RAT was analyzed by sitedirected mutagenesis. Mutations that prevented the formation of the presumed stems resulted in loss of sacB expression. Moreover, unpaired bases in RAT sequences are specificity determinants that allow the recognition of a specific RAT by its cognate antiterminator (Aymerich and Steinmetz 1992). SacY can cause antitermination with equal efficiency at RAT sequences of both the sac and bgl classes, whereas BglG and LicT have a narrow specificity, i.e. they bind to RAT sequences of the bgl class exclusively. The affinity of the RNA-binding domain of LicT for its cognate licS RAT sequence is 400-fold higher than the affinity for the sacB RAT (Manival et al 1997; Declerck et al. 1999). Quantitative gel-shift experiments have revealed that one dimer of the SacY RBD binds to one sacB RAT molecule. Moreover, RAT is able to form an intramolecular structure that is stabilized upon binding of the RBD (Manival et al. 1997). Thus, binding of the antiterminator favors formation of the RAT secondary structure which in
turn prevents the formation of the terminator and allows transcription elongation. The structure of the RBD of SacY is the first example of a novel class of RNA-binding proteins (Manival et al. 1997; van Tilbeurgh et al. 1997). The dimeric proteins were found to be symmetrical. Recently, the crystal structure of the RNA-binding domain of LicT has also been determined. The structure of the LicT RBD is very similar to that of the SacY RBD except for a more open appearance of the side of the LicT RBD dimer that contacts the RNA. Arg-27 in the RBD of LicT is required for the highly specific interaction with the licS RAT sequence, namely, with two bases that differ from the sacB RAT (Declerck et al. 1999). Analysis of the complex between the RBD of SacY and the sacB RAT sequence revealed that the global architecture of the dimer remained unaffected. The residues K4–I6, N8–N10, G25, G27, F30 and N31 are involved in the interaction with RAT. All these residues are located at the surface on one side of the protein. Mutations in these residues in SacY as well as in corresponding residues in GlcT from B. subtilis resulted in partial or complete loss of antitermination activity (Manival et al. 1997; Langbein et al. 1999). It is interesting to note that the SacY RBD binds its RAT target as a symmetrical dimer, however, RAT is asymmetrical. This might be explained by the observation that residues from different regions in each monomer (namely, H9 and F30) form a single RNA-binding site.
Antitermination of the B. subtilis glp regulon Glycerol utilization in B. subtilis requires glycerol transport, phosphorylation and conversion to dihydroxyacetone phosphate. The enzymes involved in glycerol and glycerol 3-phosphate catabolism are induced by glycerol, and induction requires a positively acting regulator, GlpP. The genes of the B. subtilis glycerol regulon form four transcriptional units: glpP, glpFK, glpD, and glpTQ (Rutberg 1997). The leader region between the glpD transcriptional start point and the glpD structural gene contains an inverted repeat, the presence of which renders expression inducible by glycerol 3-phosphate and dependent on glpP. The functionality of the inverted repeat as a terminator element was confirmed by in vitro transcription. Thus, glpD expression is controlled by transcriptional antitermination in response to the presence of glycerol 3-phosphate (Holmberg and Rutberg 1992). Inverted repeats similar to the conditional terminator of glpD are also present in the leader regions of the glpFK and glpTQ operons. It is interesting to note that the lower regions of the stems are conserved among the three potential terminators. Thus, the same regulatory mechanism may apply to the transcriptional units of the glycerol regulon (Rutberg 1997). The positive regulator of the glp regulon, GlpP, is expressed constitutively in low amounts. Binding of GlpP to its target results in antitermination and in stabilization of the glpD mRNA (Glatz et al. 1996). In contrast to the antiterminators of the BglG family which are regulated by
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reversible phosphorylation, GlpP was proposed to be active in antitermination upon binding of glycerol 3-phosphate, the intracellular inducer of the glp regulon (Rutberg 1997).
rather than by phosphorylation or direct interaction with the inducer.
Nitrogen-source-dependent antitermination Control of the P. aeruginosa AmiR antiterminator by a ligand-sensitive protein P. aeruginosa can utilize short-chain aliphatic amides by virtue of an amidase encoded by the amiE gene. Expression of amiE is positively controlled in the presence of short amides by a regulatory protein, AmiR (Cousens et al. 1987). A transcriptional terminator was identified between the constitutive promoter and the amiE ORF. Deletion of this terminator results in constitutive amidase expression which is independent of AmiR. Thus, amiE induction is mediated by transcription antitermination (Wilson and Drew 1995). In addition to amiR, a negatively acting regulator of amidase synthesis, amiC, was identified. The AmiC protein is a member of the periplasmic binding protein family. Expression of amiC and amiR in E.coli is sufficient to confer inducible expression to the amiE promoter. The presence of functional AmiR results in constitutive amiE expression, whereas AmiC is a negative factor affecting antitermination by AmiR. AmiC binds acetamide and is thus a sensor protein that in the absence of amides binds and inactivates AmiR resulting in transcription termination in the amiE leader (Wilson et al. 1993). The structure of the AmiC protein was determined. The protein is composed of two domains with an alternating β-α-β topology. The amide binding cleft is located at the interface of the domains. Upon binding of the inducer acetamide, the conformation of AmiC changes from an open to a closed form due to the contacts of acetamide with the faces of both domains. This mechanism may account for the change in the AmiR-binding capability of AmiC (Pearl et al. 1994). Direct interaction between AmiR and the amiE leader mRNA was studied in vitro. A region of 39 nucleotides located upstream of the transcriptional terminator is sufficient for the titration of AmiR, suggesting that this region is the AmiR-binding site. The apparent AmiR target does not seem to form an alternative secondary structure as discussed above for the RAT sequences. Under noninducing conditions, AmiR forms a complex with AmiC and is inactive in antitermination. By contrast, the conformational change that occurs in AmiC upon binding of acetamide results in the release of AmiR which may then act as a transcriptional antiterminator. The crystal structure of the AmiC-AmiR complex is supports this model. AmiR is composed of an N-terminal globular domain, which is the binding site of AmiC, a central coiled-coil region, which is a dimerization interface, and a C-terminal helical domain. This latter domain was proposed to be the RNAbinding domain (O’Hara et al. 1999). Thus, regulation of aliphatic amidase in P. aeruginosa is mediated by a highly unusual signal transduction system in which the transcriptional regulator is controlled by sequestration
Klebsiella oxytoca and Klebsiella aerogenes utilize nitrate both as a terminal electron acceptor for anaerobic respiration and as a source of nitrogen. Assimilatory nitrate reductase synthesis is induced by nitrate and nitrite and repressed by ammonia. The genes involved in nitrate assimilation are encoded in the nasFEDCBA operon of K. oxytoca (Lin and Stewart 1998). An attempt to isolate mutants defective in induction of nas operon expression identified the nasR gene as a positive regulator of nas operon trancription (Goldman et al. 1994). Antitermination of the nas operon is modulated by binding of NasR to alternative RNA structures. In the absence of nitrate or nitrite, a transcription terminator forms, whereas in the presence of either ion NasR can bind to a second RNA structure and prevent termination (Chai and Stewart 1998). It is worth mentioning that the two RNA structures are not mutually exclusive. It was therefore proposed that the binding of NasR to its target may either convert RNA polymerase in a terminator-resistant form as is known for phage λ antitermination or it might prevent the formation of the terminator in an as yet unknown way (Chai and Stewart 1999).
Antitermination of anabolic genes and operons Expression of several anabolic genes and operons in different bacteria is controlled by transcriptional antitermination and involves protein-RNA interactions. As described for catabolic operons, antitermination control of anabolic processes is more widespread in gram-positive than in gram-negative bacteria. Antitermination of transcription of anabolic genes was found for the expression of the tryptophan and pyrimidine biosynthetic operons in B. subtilis. The mechanisms underlying protein-RNA interaction in the regulation of tryptophan biosynthesis have been studied very intensively and shall therefore be presented first (for recent reviews, see Babitzke and Gollnick 2001; Switzer et al. 1999).
The B. subtilis TRAP protein The genes involved in tryptophan biosynthesis are located in two operons in B. subtilis: the trpEDCFBA operon is part of a histidine and aromatic amino acid biosynthetic supraoperon whereas the trpG gene encoding a glutamine amidotransferase is located in the folic acid biosynthetic operon (Babitzke and Gollnick 2001). Tryptophan biosynthetic enzymes are synthesized only in the absence of tryptophan. The presence of a putative transcriptional terminator in the leader region of the trp operon suggested the existence of an attenuation mecha-
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Fig. 2 Modulation of transcription elongation at an anabolic (trp) operon in B. subtilis. In the absence of tryptophan, the energetically more stable antitermination (AT) structure is formed and, thus, the trp operon is expressed. In the presence of tryptophan, TRAP binds to its target sites in the antiterminator region (black squares) and prevents the formation of this structure. This allows the formation of the terminator (T) and switches off the expression of the trp operon
nism as had been observed for the E. coli trp operon. In E. coli, the formation of a leader peptide modulates the transcription termination in the trp leader. However, there is no coding region for a Trp-rich leader peptide in the control region of the B. subtilis trp operon. Thus, an alternative mechanism of transcriptional attenuation is operative (Babitzke and Gollnick 2001). Transcription of the trp operon is constitutively initiated. While transcription cannot proceed beyond the terminator in the leader region in the presence of tryptophan, full-length transcripts are formed in the absence of tryptophan indicating regulation by antitermination. Upstream of the terminator in the trp leader region there is an inverted repeat, which can form a secondary structure and prevent the formation of the terminator. While deletion of the complete antitermination/termination structure resulted in constitutive expression, partial deletion of the
presumptive antitermination structure led to a total loss of trp operon expression. Thus, the formation of an alternative secondary structure may be involved in modulating termination/antitermination of the B. subtilis trp operon (Shimotsu et al. 1986) (Fig. 2). Expression of the trp operon is constitutive in mutants resistant to methyl-tryptophan. This constitutive expression is due to the elimination of transcription termination in the trp leader in mtr mutants. The genes of the mtrAB operon encode a GTP cyclohydrolase (MtrA) and a small polypeptide of about 8 kDa (MtrB) that is similar to RegA, an RNA-binding regulatory protein of bacteriophage T4 (Babitzke and Gollnick 2001). Purified MtrB protein caused premature transcription termination in the presence of tryptophan, but not in its absence. Specific binding of MtrB to trp leader mRNA was observed and MtrB was redesignated TRAP (trp RNA-binding attenuation protein) (Otridge and Gollnick 1993). A detailed analysis of the TRAP-binding site in the trp leader mRNA identified 11 closely spaced GAG and UAG repeats as TRAP targets. Similarly spaced trinucleotide repeats are also present surrounding the ribosomal-binding site of the trpG gene which is also controlled by TRAP. The solution of the crystal structure of TRAP revealed that the protein is composed of 11 subunits and that it exhibits an 11-fold symmetry (Antson et al. 1995). This indicates an interaction of each monomer with one of the 11 trinucleotide repeats in the trp leader (Babitzke et al. 1994) (Fig. 2). Optimal TRAP-binding sites contain multiple G/UAG repeats that are separated by two to three nucleotides and a stem-loop structure upstream of the actual TRAP-binding site. The formation of this structure is required for proper TRAP-dependent transcriptional attenuation of the trp operon (Sudershana et al. 1999). The solution of the structures of TRAP and its complex with the trp leader mRNA revealed that the RNA is wrapped around the TRAP 11-mer (Antson et al. 1999) (Fig. 2). The arrangement of the 11 subunits of TRAP is stabilized by inter-subunit β-sheets. The 11-mer forms a β-wheel with a large central hole. Interestingly, TRAP does not contain any α-helical elements (Antson et al. 1995). Three basic amino acids in TRAP are important for RNA binding, these are K37, K56, and R58. It was therefore proposed that RNA wraps around TRAP by interacting with these residues (Antson et al. 1999). TRAP activity is controlled by direct binding of tryptophan and by interaction with the TRAP-inhibitory protein AT. Tryptophan binding occurs in clefts between adjacent β-sheets. Binding of one tryptophan molecule per subunit is a highly cooperative process resulting in a conformational change in the flexible residues between the strands β2/β3 and β4/β5 (Antson et al. 1995). In the absence of tryptophan uncharged tRNATrp allows expression of the AT protein which binds TRAP to inhibit its activity. Thus, in the presence of tryptophan, TRAP is activated and will thus prevent trp operon transcription, whereas TRAP is inactivated by AT in the absence of tryptophan resulting in expression of the trp operon (Valbuzzi and Yanofski 2001).
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TRAP binding to the trp leader mRNA has two regulatory consequences: (1) as discussed above, formation of the antiterminator structure is inhibited, thus causing premature transcription termination; (2) a secondary structure may be formed that blocks the access of ribosomes to the ribosomal binding site of trpE, the first gene of the operon. In contrast to the regulation of the trp operon by TRAP-mediated antitermination/termination control, expression of the trpG gene is regulated by TRAP exclusively at the level of translation (Babitzke and Gollnick 2001).
The B. subtilis PyrR protein – an enzyme able to control gene expression by RNA binding The genes required for pyrimidine biosynthesis are encoded in the pyr operon in B. subtilis. Expression of this operon is repressed by pyrimidines. The promoter of the operon is constitutively active and a potential terminator is located immediately downstream of the transcription start point. Deletion of this terminator abolishes regulation of the pyr operon by pyrimidines. Thus, the B. subtilis pyr operon is controlled by transcriptional antitermination (Quinn et al. 1991). In addition to the terminator in the leader, there are two other potential terminators located in the pyrR-pyrP and pyrP-pyrB intercistronic regions (pyrR is the first gene of the ten-gene operon). All of these terminators are active in a pyrimidine-dependent fashion. The terminators are preceded by another stemloop structure, which might contain RAT sequences. In their promoter-proximal half, the potential RAT sites have a conserved motif of 50 nucleotides which may be the binding site for a regulatory protein. Deletion of the first gene of the pyr operon, pyrR, results in constitutive expression of the operon suggesting that PyrR is the regulator. In addition to its regulatory function, PyrR acts also as a minor uracil phosphoribosyltransferase. In the presence of pyrimidines PyrR binds to the conserved sites in the potential RAT structures thus preventing the formation of these structures and allowing terminator formation (Turner et al. 1994). Similar regulatory mechanisms were also suggested for Bacillus caldolyticus, Enterococcus faecalis, Lactocbacillus plantarum and Lactococcus lactis. Regulation by PyrR is not restricted to gram-positive bacteria: a Thermus sp. encodes a PyrR that probably binds to pyr mRNA to act as a translational repressor (similar to the action of TRAP at trpG) (Switzer et al. 1999). The three antiterminator/terminator regions in the B. subtilis pyr operon confer individually a three- to fivefold regulation to the pyr operon, while together they act in a cumulative manner. Regulation at all three terminators depends on PyrR. In a pyrR mutant, the terminators are not active probably because the energetically more stable RAT structures form in such a mutant (Switzer et al. 1999). Mutations resulting in constitutive pyr expression have been isolated in an attempt to define the cis-acting sites involved in regulation of the operon. Out of 12
different mutations studied, two were located in the terminator and might therefore prevent the proper transcription termination in the presence of pyrimidines. Ten mutations were clustered in the promoter-proximal part of the antiterminator structure that is conserved in the three attenuation sites of the B. subtilis pyr operon and was proposed to be the PyrR-binding site. It is interesting to note that no repeated sequences such as the binding site for TRAP are present in the pyr control regions. Thus, the mechanism of PyrR-RNA recognition and binding seems to differ substantially from the one described for TRAP (see above) (Ghim and Switzer 1996). In vitro transcriptional analyses using antisense oligonucleotides to disrupt the secondary structure revealed the existence of a structure in addition to the terminator and the antiterminator. This structure, designated the anti-antiterminator, is located upstream of the antiterminator. Formation of the anti-antiterminator is energetically unfavorable. However, the formation of the anti-antiterminator and the antiterminator structures is mutually exclusive. Based on the data obtained from cisactive mutations described above, it was concluded that PyrR might bind to and thus stabilize the anti-antiterminator in the presence of pyrimidines. This would result in formation of the terminator. By contrast, in the absence of pyrimidines, the antiterminator can form and thus enable expression of the pyr operon (Lu et al. 1996). The activity of PyrR is controlled by the availability of pyrimidine nucleotides. Termination of transcription due to PyrR binding to the anti-antiterminator is most efficient in the presence of UMP, whereas UTP and UDP are much less effective in causing PyrR-dependent transcription termination. Besides its role in controlling the expression of the pyr operon, PyrR has also an uracil phosphoribosyltransferase activity. Phosphoribosylpyrophosphate, the second substrate of this enzymatic activity, is a strong antagonist of UMP in the control of PyrR activity. This might result from the competition between the two compounds for binding to the same site in PyrR (Lu and Switzer 1996). Purified PyrR exhibits enzymatic activity and binds the pyr operon leader mRNA in vitro. Binding to the RNA is specific and requires the presence of UMP (Turner et al. 1998). The crystal structure of PyrR is similar to that of other phosphoribosyltransferases (PRTases). However, the dimer is different from other PRTases. A large basic surface at the dimer interface may serve as an RNA-binding site. These results were interpreted as an indication for the evolution of an enzyme towards a novel, regulatory, function (Tomchick et al. 1998). Interestingly, this is paralleled by aconitase from B. subtilis and other bacteria which is also active as an iron-dependent enzyme in the tricarboxylic acid cycle and alternatively as a RNAbinding protein in the absence of iron (Alén and Sonenshein 1999).
Concluding remarks Transcriptional antitermination is probably much more widespread than previously anticipated. Even though pro-
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tein sequences of complete genomes are easily handled these days, new proteins continue to turn out to be RNAbinding proteins modulating transcription termination/antitermination, with the most recent representative being the CspA family of RNA chaperones and the proposed antitermination system of sulfonate/sulfur utilization in B. subtilis (van der Ploeg et al. 2001). This reflects the structural diversity of protein-RNA interaction as compared to protein-DNA interaction. Moreover, the activity of the RNA-binding regulatory protein can be controlled in several distinct ways such as direct interaction with an inducer, phosphorylation or interaction with a sensor protein. Their is no doubt that even more interesting examples of regulatory protein-RNA interactions remain to be uncovered.
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