Transcription Attenuation Mechanism Research Articles

Folding of the lysine riboswitch: importance of peripheral elements for transcriptional regulation

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2–loop L3 and helix P2–loop L4)—all of this for the creation of a specific lysine binding site. We have determined that the interaction P2–L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2–L3 and P2–L4 allows both interactions to fold at lower magnesium ion concentrations. The P2–L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem–loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation.

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism controlled by the trp RNA-binding attenuation protein (TRAP). TRAP binds to 11 (G/U)AG repeats in the trp leader transcript and prevents formation of an antiterminator, which allows formation of an intrinsic terminator (attenuator). Previously, formation of the attenuator RNA structure was believed to be solely responsible for signaling RNA polymerase (RNAP) to halt transcription. However, base substitutions that prevent formation of the antiterminator, and thus allow the attenuator structure to form constitutively, do not result in efficient transcription termination. The observation that the attenuator requires the presence of TRAP bound to the nascent RNA to cause efficient transcription termination suggests TRAP has an additional role in causing termination at the attenuator. We show that the trp attenuator is a weak intrinsic terminator due to low GC content of the hairpin stem and interruptions in the U-stretch following the hairpin. We also provide evidence that termination at the trp attenuator requires forward translocation of RNA polymerase and that TRAP binding to the nascent transcript can induce this activity.

Mechanism of NusG-stimulated pausing, hairpin-dependent pause site selection and intrinsic termination at overlapping pause and termination sites in the Bacillus subtilis trp leader

The Bacillus subtilis trpEDCFBA operon is regulated by TRAP-dependent transcription attenuation and translation repression mechanisms. Previous results showed that NusA and NusG cooperatively stimulate RNA polymerase pausing at U107 and U144 in the trp leader, and that NusG is required for pausing at U144 in vivo. Pausing at U107 and U144 participate in the attenuation and translation repression mechanisms, respectively, by providing additional time for TRAP binding. The intrinsic trp leader terminator overlaps the hairpin-dependent U144 pause site. Here, we conducted a systematic mutational analysis of the terminator/pause region. Deletion of the hairpin reduced pausing but did not affect pause site selection. Thus, hairpin-stimulated pausing is a more appropriate term than hairpin-dependent pausing for this pause site. In contrast, minor changes to the hairpin abolished termination. Sequences in the U-rich/T-rich tract following the hairpin affected termination and pausing differentially. The distance between the hairpin and the 3' end of the RNA dictates the position of termination, whereas the sequence downstream from the hairpin is responsible for pause site selection. NusA was found to increase both pausing and termination by reducing the rate of transcription. We also found that NusG-stimulated pausing is sequence specific and that NusG does not affect termination.

A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri.

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation.

Transcriptional regulation of histidine biosynthesis genes in Corynebacterium glutamicum

Corynebacterium glutamicum, a gram-positive bacterium, has been widely used for industrial amino acid production. Corynebacterium glutamicum his genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. The latter his operon starts the transcription at the C residue localized 196 bp upstream of the hisD ATG start codon. Our computer-based sequence analysis showed that the region corresponding to the untranslated 5' end of the transcript, named the hisD leader region, displays the typical features of the T-box transcriptional attenuation mechanism. Therefore, expression of the cat reporter gene under the control of the wild-type or mutated hisD leader regions was tested in multi-copy (pProm and pTer series) and in single-copy (pInt series) systems under conditions of sufficient or limited histidine. Our mutational studies led to the conclusion that the CAU histidine specifier and 5'-UGGA-3' sequence in the hisD leader region are required for the hisDCB-orf1-orf2-hisHA-impA-hisFI gene regulation. The cat gene expression from the wild-type leader region was negatively regulated by histidine. However, the cat gene expression from mutated leader regions was irresponsive to the level of histidine in the growth medium. Taken together, we propose that a T-box mediated attenuation mechanism is responsible for the gene expression of the hisDCB-orf1-orf2-hisHA-impA-hisFI operon in C. glutamicum.

Biochemical Features and Functional Implications of the RNA-Based T-Box Regulatory Mechanism

The T-box mechanism is a common regulatory strategy used for modulating the expression of genes of amino acid metabolism-related operons in gram-positive bacteria, especially members of the Firmicutes. T-box regulation is usually based on a transcription attenuation mechanism in which an interaction between a specific uncharged tRNA and the 5' region of the transcript stabilizes an antiterminator structure in preference to a terminator structure, thereby preventing transcription termination. Although single T-box regulatory elements are common, double or triple T-box arrangements are also observed, expanding the regulatory range of these elements. In the present study, we predict the functional implications of T-box regulation in genes encoding aminoacyl-tRNA synthetases, proteins of amino acid biosynthetic pathways, transporters, and regulatory proteins. We also consider the global impact of the use of this regulatory mechanism on cell physiology. Novel biochemical relationships between regulated genes and their corresponding metabolic pathways were revealed. Some of the genes identified, such as the quorum-sensing gene luxS, in members of the Lactobacillaceae were not previously predicted to be regulated by the T-box mechanism. Our analyses also predict an imbalance in tRNA sensing during the regulation of operons containing multiple aminoacyl-tRNA synthetase genes or biosynthetic genes involved in pathways common to more than one amino acid. Based on the distribution of T-box regulatory elements, we propose that this regulatory mechanism originated in a common ancestor of members of the Firmicutes, Chloroflexi, Deinococcus-Thermus group, and Actinobacteria and was transferred into the Deltaproteobacteria by horizontal gene transfer.

Function of the Bacillus subtilis transcription elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader

NusA and NusG are transcription elongation factors that bind to RNA polymerase (RNAP) after sigma subunit release. Escherichia coli NusA (NusA(Ec)) stimulates intrinsic termination and RNAP(Ec) pausing, whereas NusG(Ec) promotes Rho-dependent termination and pause escape. Both Nus factors also participate in the formation of RNAP(Ec) antitermination complexes. We showed that Bacillus subtilis NusA (NusA(Bs)) stimulates intrinsic termination and RNAP(Bs) pausing at U107 and U144 in the trpEDCFBA operon leader. Pausing at U107 and U144 participates in the transcription attenuation and translational control mechanisms, respectively, presumably by providing additional time for trp RNA-binding attenuation protein (TRAP) to bind to the nascent trp leader transcript. Here, we show that NusG(Bs) causes modest pause stimulation at U107 and dramatic pause stimulation at U144. NusA(Bs) and NusG(Bs) act synergistically to increase the U107 and U144 pause half-lives. NusG(Bs)-stimulated pausing at U144 requires RNAP(Bs), whereas NusA(Bs) stimulates pausing of RNAP(Bs) and RNAP(Ec) at the U144 and E. coli his pause sites. Although NusG(Ec) does not stimulate pausing at U144, it competes with NusG(Bs)-stimulated pausing, suggesting that both proteins bind to the same surface of RNAP(Bs). Inactivation of nusG results in the loss of RNAP pausing at U144 in vivo and elevated trp operon expression, whereas plasmid-encoded NusG complements the mutant defects. Overexpression of nusG reduces trp operon expression to a larger extent than overexpression of nusA.

TRAP-5′ stem–loop interaction increases the efficiency of transcription termination in the Bacillus subtilis trpEDCFBA operon leader region

TRAP regulates expression of the Bacillus subtilis trpEDCFBA operon by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the nascent trp leader transcript. Bound TRAP blocks formation of an antiterminator structure and allows formation of an overlapping intrinsic terminator upstream of the trp operon structural genes. A 5' stem-loop (5'SL) structure located upstream of the triplet repeat region also interacts with TRAP. TRAP-5'SL RNA interaction participates in the transcription attenuation mechanism by preferentially increasing the affinity of TRAP for the nascent trp leader transcript during the early stages of transcription, when only a few triplet repeats have been synthesized. Footprinting assays indicated that the 5'SL contacts TRAP through two discrete groups of single-stranded nucleotides that lie in the hairpin loop and in an internal loop. Filter binding and in vivo expression assays of 5'SL mutants established that G7, A8, and A9 from the internal loop, and A19 and G20 from the hairpin loop are critical for proper 5'SL function. These nucleotides are conserved among certain other 5'SL-containing organisms. Single-round transcription results indicated that the 5'SL increases the termination efficiency when transcription is fast; however, the influence of the 5'SL was lost when transcription was slowed by reducing the ribonucleoside triphosphate concentration. Since there is a limited amount of time for TRAP to bind to the nascent transcript and promote termination, our data suggest that the contribution of TRAP-5'SL interaction increases the rate of TRAP binding, which, in turn, increases the efficiency of transcription termination.

Regulation of pyr Gene Expression in Mycobacterium smegmatis by PyrR-Dependent Translational Repression

Regulation of pyrimidine biosynthetic (pyr) genes by a transcription attenuation mechanism that is mediated by the PyrR mRNA-binding regulatory protein has been demonstrated for numerous gram-positive bacteria. Mycobacterial genomes specify pyrR genes and contain obvious PyrR-binding sequences in the initially transcribed regions of their pyr operons, but transcription antiterminator and attenuation terminator sequences are absent from their pyr 5' leader regions. This work demonstrates that repression of pyr operon expression in Mycobacterium smegmatis by exogenous uracil requires the pyrR gene and the pyr leader RNA sequence for binding of PyrR. Plasmids containing the M. smegmatis pyr promoter-leader region translationally fused to lacZ also displayed pyrR-dependent repression, but transcriptional fusions of the same sequences to a lacZ gene that retained the lacZ ribosome-binding site were not regulated by PyrR plus uracil. We propose that PyrR regulates pyr expression in M. smegmatis, other mycobacteria, and probably in numerous other bacteria by a translational repression mechanism in which nucleotide-regulated binding of PyrR occludes the first ribosome-binding site of the pyr operon.

The Rate of TRAP Binding to RNA Is Crucial for Transcription Attenuation Control of the B. subtilis trp Operon

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic and transport genes in Bacillus subtilis in response to changes in the levels of intracellular tryptophan. Transcription of the trpEDCFBA operon is controlled by an attenuation mechanism involving two overlapping RNA secondary structures in the 5′ leader region of the trp transcript; TRAP binding promotes formation of a transcription terminator structure that halts transcription prior to the structural genes. TRAP consists of 11 identical subunits and is activated to bind RNA by binding up to 11 molecules of L-tryptophan. The TRAP binding site in the leader region of the trp operon mRNA consists of 11 (G/U)AG repeats. We examined the importance of the rate of TRAP binding to RNA for the transcription attenuation mechanism. We compared the properties of two types of TRAP 11-mers: homo-11-mers composed of 11 wild-type subunits, and hetero-11-mers with only one wild-type subunit and ten mutant subunits defective in binding either RNA or tryptophan. The hetero-11-mers bound RNA with only slightly diminished equilibrium binding affinity but with slower on-rates as compared to WT TRAP. The hetero-11-mers showed significantly decreased ability to induce transcription termination in the trp leader region when examined using an in vitro attenuation system. Together these results indicate that the rate of TRAP binding to RNA is a crucial factor in TRAP's ability to control attenuation.

The regulator protein PyrR of Bacillus subtilis specifically interacts in vivo with three untranslated regions within pyr mRNA of pyrimidine biosynthesis

In vitro experiments have shown that the genes of the de novo pyrimidine biosynthetic pathway of Bacillus subtilis, the pyr genes, are regulated by a transcriptional attenuation mechanism. Specific regulatory sequences (binding loops, BLs) are located within three untranslated leader sequences at the beginning of pyr mRNA. These binding loops, BL1, BL2 and BL3, act as anti-antiterminators of transcription when stabilized by the regulator protein PyrR. In this work, the interaction of PyrR with BL1, BL2 and BL3 was qualitatively and quantitatively analysed in vivo using the yeast three-hybrid system. The results indicate that PyrR specifically binds to BL1, BL2 and BL3. Furthermore, the data suggest that the strength of interaction between PyrR and the three different BLs in vivo is within the same dimension. The yeast three-hybrid system also proved to be useful for the rapid analysis of structural requirements for PyrR-BL binding. Point mutations within the predicted critical regions of BL1, BL2 and BL3 led to drastically reduced binding of PyrR. In summary, it is shown that the yeast three-hybrid system is well suited to qualitatively and quantitatively analyse bacterial regulatory systems that are based on factor-independent transcriptional attenuation.

Ribosomal protein L20 controls expression of the Bacillus subtilis infC operon via a transcription attenuation mechanism

In contrast to Escherichia coli no molecular mechanism controlling the biosynthesis of ribosomal proteins has been elucidated in Gram-positive organisms. Here we show that the expression of the Bacillus subtilis infC-rpmI-rplT operon encoding translation factor IF3 and the ribosomal proteins L35 and L20 is autoregulated by a complex transcription attenuation mechanism. It implicates a 200-bp leader region upstream of infC which contains two conserved regulatory elements, one of which can act as a transcription terminator. Using in vitro and in vivo approaches we show that expression of the operon is regulated at the level of transcription elongation by a change in the structure of the leader mRNA which depends upon the presence of ribosomal protein L20. L20 binds to a phylogenetically conserved domain and provokes premature transcription termination at the leader terminator. Footprint and toeprint experiments support a regulatory model involving molecular mimicry between the L20-binding sites on 23S rRNA and the mRNA. Our data suggest that Nomura's model of ribosomal protein biosynthesis based on autogenous control and molecular mimicry is also valid in Gram-positive organisms.

The trp RNA‐binding attenuation protein (TRAP) of Bacillus subtilis regulates translation initiation of ycbK, a gene encoding a putative efflux protein, by blocking ribosome binding

Expression of the Bacillus subtilis tryptophan biosynthetic genes trpEDCFBA and trpG, as well as a putative tryptophan transport gene (trpP), are regulated in response to tryptophan by the trp RNA-binding attenuation protein (TRAP). TRAP regulates expression of these genes by transcription attenuation and translation control mechanisms. Here we show that TRAP also regulates translation of ycbK, a gene that encodes a protein with similarities to known efflux proteins. As a likely TRAP-binding site consisting of 11 NAG repeats overlaps the ycbK translation initiation region, experiments were carried out to determine whether TRAP regulates translation of ycbK. TRAP was observed to regulate expression of a ycbK'-'lacZ translational fusion 20-fold in response to tryptophan. Binding studies indicated that TRAP binds to the ycbK transcript with high affinity and specificity. Footprint studies revealed that the central seven triplet repeats were protected by bound TRAP, while toeprint results suggest that nine triplet repeats contribute to TRAP binding. Additional toeprint and in vitro translation analyses demonstrated that bound TRAP regulates YcbK synthesis by blocking ribosome binding. We also identified two dipeptide coding minigenes between the Shine-Dalgarno sequence and start codon of ycbK. Expression of one of the minigenes modestly interfered with translation of ycbK.

Identification of small Hfq-binding RNAs in Listeria monocytogenes

The RNA-binding protein Hfq plays important roles in bacterial physiology and is required for the activity of many small regulatory RNAs in prokaryotes. We have previously shown that Hfq contributes to stress tolerance and virulence in the Gram-positive human pathogen Listeria monocytogenes. In the present study, we performed coimmunoprecipitations followed by enzymatic RNA sequencing to identify Hfq-binding RNA molecules in L. monocytogenes. The approach resulted in the discovery of three small RNAs (sRNAs). The sRNAs are conserved between Listeria species, but were not identified in other bacterial species. The initial characterization revealed a number of unique features displayed by each individual sRNA. The first sRNA is encoded from within an annotated gene in the L. monocytogenes EGD-e genome. Analogous to most regulatory sRNAs in Escherichia coli, the stability of this sRNA is highly dependent on the presence of Hfq. The second sRNA appears to be produced by a transcription attenuation mechanism, and the third sRNA is present in five copies at two different locations within the L. monocytogenes EGD-e genome. The cellular levels of the sRNAs are growth phase dependent and vary in response to growth medium. All three sRNAs are expressed when L. monocytogenes multiplies within mammalian cells. This study represents the first attempt to identify sRNAs in L. monocytogenes.

Bacillus subtilis TRAP Binds to its RNA Target by a 5′ to 3′ Directional Mechanism

TRAP is an 11 subunit RNA-binding protein that regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms. Tryptophan-activated TRAP acts by binding to a site in the 5'-untranslated leader region of trp mRNA consisting of 11 (G/U)AG repeats. We used mung bean nuclease footprinting to analyze the interaction of TRAP with several artificial binding sites composed of 11 GAG repeats in nucleic acids that lack secondary structure. Affinities for individual repeats within a binding site did not vary significantly. In contrast, the association rate constants were highest for repeats at the 5' end and lowest for those at the 3' end of all binding sites tested. These results indicate that TRAP binds to its RNA targets by first associating with one or more repeat at the 5' end of its binding site followed by wrapping the remainder of binding site around the protein in a 5' to 3' direction. This directional binding is novel among RNA-binding proteins. We suggest that this mechanism of binding is important for TRAP-mediated transcription attenuation control of the trp operon.

Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus.

Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. We recently determined that the B. subtilis trp leader readthrough transcript can adopt a Mg(2+)-dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE. In the present study, sequence comparisons to trp leaders from three other Bacillus sp. were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved. To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus. Structure mapping experiments confirmed the predicted secondary structure. Native gel experiments identified a faster mobility species in the presence of Mg(2+), suggesting that a Mg(2+)-dependent tertiary structure forms. Mg(2+)-dependent protection of residues within the first five triplet repeats of the TRAP binding target and a pyrimidine-rich internal loop were observed, consistent with tertiary structure formation between these regions. Structure mapping in the presence of a competitor DNA oligonucleotide allowed the interacting partners to be identified as a single-stranded portion of the purine-rich TRAP binding target and the large downstream pyrimidine-rich internal loop. Thermal denaturation experiments revealed a Mg(2+)- and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats. The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined. These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.

A Mg2+-dependent RNA Tertiary Structure Forms in the Bacillus subtilis trp Operon Leader Transcript and Appears to Interfere with trpE Translation Control by Inhibiting TRAP Binding

Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure. We examined the structure of the trp leader readthrough RNA in the absence of TRAP. Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models. In addition, this RNA was found to adopt a Mg2+-dependent, long-range tertiary interaction under physiological monovalent salt conditions. Formation of this tertiary structure does not require significant changes in the preformed secondary structure. Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop. UV cross-linking experiments utilizing 5′-p-azidophenacyl-containing transcripts revealed a Mg2+-dependent cross-link. Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop. Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure. Filter-binding studies demonstrated that the addition of Mg2+ inhibits TRAP binding, which may be partially due to the effect of Mg2+ on RNA tertiary structure formation. Results from expression studies using trpE′-′lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg2+-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE.

Role of RNA Structure in Transcription Attenuation in Bacillus subtilis: The trpEDCFBA Operon as a Model System

Publisher Summary This chapter describes methods to examine the role of RNA structure plays in transcription attenuation, using studies Bacillus subtilis trpEDCFBA operon as a model system. Transcription attenuation mechanisms modulate the extent of transcription read-through past termination signals, thereby regulating expression of the downstream genes. The untranslated trp leader transcript can form four RNA secondary structures that participate in the attenuation mechanism: the 5ʹ stem-loop, the pause hairpin, the antiterminator, and the terminator. RNA secondary structure can be predicted by comparative sequence analysis if reliable secondary structures already exist for several homologues, or it can be predicted by folding programs such as mfold, which rely on free energy minimization. The chapter discusses the biochemical determination of RNA secondary structure. Once formation of an RNA secondary structure is verified biochemically, it is necessary to establish that the structure participates in transcription attenuation. This can be accomplished through the use of genetic and biochemical approaches. Two different in vitro transcription assays are generally used to determine if an RNA structure participates in transcription attenuation.

Comparative Genomics of Thiamin Biosynthesis in Procaryotes: NEW GENES AND REGULATORY MECHANISMS

Vitamin B(1) in its active form thiamin pyrophosphate is an essential coenzyme that is synthesized by coupling of pyrimidine (hydroxymethylpyrimidine; HMP) and thiazole (hydroxyethylthiazole) moieties in bacteria. Using comparative analysis of genes, operons, and regulatory elements, we describe the thiamin biosynthetic pathway in available bacterial genomes. The previously detected thiamin-regulatory element, thi box (Miranda-Rios, J., Navarro, M., and Soberon, M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9736-9741), was extended, resulting in a new, highly conserved RNA secondary structure, the THI element, which is widely distributed in eubacteria and also occurs in some archaea. Search for THI elements and analysis of operon structures identified a large number of new candidate thiamin-regulated genes, mostly transporters, in various prokaryotic organisms. In particular, we assign the thiamin transporter function to yuaJ in the Bacillus/Clostridium group and the HMP transporter function to an ABC transporter thiXYZ in some proteobacteria and firmicutes. By analogy to the model of regulation of the riboflavin biosynthesis, we suggest thiamin-mediated regulation based on formation of alternative RNA structures involving the THI element. Either transcriptional or translational attenuation mechanism may operate in different taxonomic groups, dependent on the existence of putative hairpins that either act as transcriptional terminators or sequester translation initiation sites. Based on analysis of co-occurrence of the thiamin biosynthetic genes in complete genomes, we predict that eubacteria, archaea, and eukaryota have different pathways for the HMP and hydroxyethylthiazole biosynthesis.

Sensing Small Molecules by Nascent RNA: A Mechanism to Control Transcription in Bacteria

Thiamin and riboflavin are precursors of essential coenzymes—thiamin pyrophosphate (TPP) and flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD), respectively. In Bacillus spp, genes responsible for thiamin and riboflavin biosynthesis are organized in tightly controllable operons. Here, we demonstrate that the feedback regulation of riboflavin and thiamin genes relies on a novel transcription attenuation mechanism. A unique feature of this mechanism is the formation of specific complexes between a conserved leader region of the cognate RNA and FMN or TPP. In each case, the complex allows the termination hairpin to form and interrupt transcription prematurely. Thus, sensing small molecules by nascent RNA controls transcription elongation of riboflavin and thiamin operons and possibly other bacterial operons as well.