The THI-box riboswitch, or how RNA binds thiamin pyrophosphate

Riboswitches are genetic control elements present mainly in the 5' untranslated regions of messenger RNAs that, upon binding of a small metabolite (like some vitamins, amino acids, and nucleobases), undergo conformational changes, affecting the expression of downstream genes. Structural studies of riboswitches are important for understanding how they recognize their ligands with high specificity and affinity. The thiamin pyrophosphate binding riboswitch (THI- box) is widely distributed in the three kingdoms of life and is involved in very distinct modes of gene regulation. Three recent THI-box structural analyses revealed how polyanionic RNA is able to bind a molecule with a negatively charged pyrophosphate group like thiamin pyrophosphate (TPP) and how it can discriminate between TPP and monophosphorylated analog molecules. These studies give insight into the genetic regulatory mechanisms in which the THI-box is involved.     

RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ~7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding.     

Regulation of gene expression by effectors that bind to RNA

Recent studies have revealed several genetic systems in bacteria that use complex RNA structural elements to monitor regulatory signals and control expression of downstream genes. These include RNA thermosensors, in which an inhibitory structure melts at high temperature, and systems where binding of small RNAs or cellular metabolites modulates the structure of the RNA. The remarkable feature of these systems is the ability of the regulatory RNA elements to specifically sense the regulatory signal, without accessory components, and convey that information to the gene expression machinery via a structural change in the nascent RNA.     

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.     

Ligand binding and gene control characteristics of tandem riboswitches in Bacillus anthracis

Most riboswitches are composed of a single metabolite-binding aptamer and a single expression platform that function together to regulate genes in response to changing metabolite concentrations. In rare instances, two aptamers or sometimes two complete riboswitches reside adjacent to each other in untranslated regions (UTRs) of mRNAs. We have examined an example of a tandem riboswitch in the Gram-positive bacterium Bacillus anthracis that includes two complete riboswitches for thiamine pyrophosphate (TPP). Unlike other complex riboswitch systems described recently, tandem TPP riboswitches do not exhibit cooperative ligand binding and do not detect two different types of metabolites. In contrast, both riboswitches respond independently to TPP and are predicted to function in concert to mimic the more "digital" gene control outcome observed when two aptamers bind ligands cooperatively. Our findings further demonstrate that simple gene control elements made only of RNA can be assembled in different architectures to yield more complex gene control outcomes.     

Ligand-dependent folding of the three-way junction in the purine riboswitch

Riboswitches are highly structured cis-acting elements located in the 5'-untranslated region of messenger RNAs that directly bind small molecule metabolites to regulate gene expression. Structural and biochemical studies have revealed riboswitches experience significant ligand-dependent conformational changes that are coupled to regulation. To monitor the coupling of ligand binding and RNA folding within the aptamer domain of the purine riboswitch, we have chemically probed the RNA with N-methylisatoic anhydride (NMIA) over a broad temperature range. Analysis of the temperature-dependent reactivity of the RNA in the presence and absence of hypoxanthine reveals that a limited set of nucleotides within the binding pocket change their conformation in response to ligand binding. Our data demonstrate that a distal loop-loop interaction serves to restrict the conformational freedom of a significant portion of the three-way junction, thereby promoting ligand binding under physiological conditions.     

The crystal structure of yeast thiamin pyrophosphokinase.

BACKGROUND: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyrophosphate group from ATP to vitamin B1 (thiamin) to form the coenzyme thiamin pyrophosphate (TPP). Thus, TPK is important for the formation of a coenzyme required for central metabolic functions. TPK has no sequence homologs in the PDB and functions by an unknown mechanism. The TPK structure has been determined as a significant step toward elucidating its catalytic action. RESULTS: The crystal structure of Saccharomyces cerevisiae TPK complexed with thiamin has been determined at 1.8 A resolution. TPK is a homodimer, and each subunit consists of two domains. One domain resembles a Rossman fold with four alpha helices on each side of a 6 strand parallel beta sheet. The other domain has one 4 strand and one 6 strand antiparallel beta sheet, which form a flattened sandwich structure containing a jelly-roll topology. The active site is located in a cleft at the dimer interface and is formed from residues from domains of both subunits. The TPK dimer contains two compound active sites at the subunit interface. CONCLUSIONS: The structure of TPK with one substrate bound identifies the location of the thiamin binding site and probable catalytic residues. The structure also suggests a likely binding site for ATP. These findings are further supported by TPK sequence homologies. Although possessing no significant sequence homology with other pyrophospokinases, thiamin pyrophosphokinase may operate by a mechanism of pyrophosphoryl transfer similar to those described for pyrophosphokinases functioning in nucleotide biosynthesis.     

Conformational changes in the expression domain of the Escherichia coli thiM riboswitch

The thiM riboswitch contains an aptamer domain that adaptively binds the coenzyme thiamine pyrophosphate (TPP). The binding of TPP to the aptamer domain induces structural rearrangements that are relayed to a second domain, the so-called expression domain, thereby interfering with gene expression. The recently solved crystal structures of the aptamer domains of the thiM riboswitches in complex with TPP revealed how TPP stabilizes secondary and tertiary structures in the RNA ligand complex. To understand the global modes of reorganization between the two domains upon metabolite binding the structure of the entire riboswitch in presence and absence of TPP needs to be determined. Here we report the secondary structure of the entire thiM riboswitch from Escherichia coli in its TPP-free form and its transition into the TPP-bound variant, thereby depicting domains of the riboswitch that serve as communication links between the aptamer and the expression domain. Furthermore, structural probing provides an explanation for the lack of genetic control exerted by a riboswitch variant with mutations in the expression domain that still binds TPP.     

Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells

The thiamin transporter encoded by SLC19A2 and the reduced folate carrier (RFC1) share 40% homology at the protein level, but the thiamin transporter does not mediate transport of folates. By using murine leukemia cell lines that express no, normal, or high levels of RFC1, we demonstrate that RFC1 does not mediate thiamin influx. However, high level RFC1 expression substantially reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized intracellularly from imported thiamin, resulted from RFC1-mediated efflux of TPP. This conclusion was supported by the following observations. (i) Efflux of intracellular TPP was increased in cells with high expression of RFC1. (ii) Methotrexate inhibits TPP influx. (iii) TPP competitively inhibits methotrexate influx. (iv) Loading cells, which overexpress RFC1 to high levels of methotrexate to inhibit competitively RFC1-mediated TPP efflux, augment TPP accumulation. (v) There was an inverse correlation between thiamin accumulation and RFC1 activity in cells grown at a physiological concentration of thiamin. The modulation of thiamin accumulation by RFC1 in murine leukemia cells suggests that this carrier may play a role in thiamin homeostasis and could serve as a modifying factor in thiamin nutritional deficiency as well as when the high affinity thiamin transporter is mutated.     

Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis

Riboswitches are structured mRNA elements that regulate gene expression upon binding specific cellular metabolites. It is thought that the highly conserved metabolite-binding domains of riboswitches undergo conformational change upon binding their cognate ligands. To investigate the generality of such a mechanism, we employed small-angle X-ray scattering (SAXS). We probed the nature of the global metabolite-induced response of the metabolite-binding domains of four different riboswitches that bind, respectively, thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), lysine, and S-adenosyl methionine (SAM). We find that each RNA is unique in its global structural response to metabolite. Whereas some RNAs exhibit distinct free and bound conformations, others are globally insensitive to the presence of metabolite. Thus, a global conformational change of the metabolite-binding domain is not a requirement for riboswitch function. It is possible that the range of behaviors observed by SAXS, rather than being a biophysical idiosyncrasy, reflects adaptation of riboswitches to the regulatory requirements of their individual genomic context.     

Riboswitches as versatile gene control elements

Riboswitches are structured elements typically found in the 5' untranslated regions of mRNAs, where they regulate gene expression by binding to small metabolites. In all examples studied to date, these RNA control elements do not require the involvement of protein factors for metabolite binding. Riboswitches appear to be pervasive in eubacteria, suggesting that this form of regulation is an important mechanism by which metabolic genes are controlled. Recently discovered riboswitch classes have surprisingly complex mechanisms for regulating gene expression and new high-resolution structural models of these RNAs provide insight into the molecular details of metabolite recognition by natural RNA aptamers.     

piR_015520 belongs to Piwi-associated RNAs regulates expression of the human melatonin receptor 1A gene

Piwi-associated RNAs (piRNAs) are a distinct class of 24- to 30-nucleotide-long RNAs produced by a Dicer-independent mechanism, and are associated with Piwi-class Argonaute proteins. In contrast to the several hundred species of microRNAs (miRNAs) identified thus far, piRNAs consist of more than 30,000 different species in humans. Studies in flies, fish and mice implicate these piRNAs in regulating germ line development, the silencing of selfish DNA elements, and maintaining germ line DNA integrity. Most piRNAs map to unique sites in the human genome, including intergenic, intronic, and exonic sequences. However, the role of piRNAs in humans remains to be elucidated. Here, we uncover an unexpected function of the piRNA pathway in humans. We show for the first time, that the piRNA_015520, located in intron 1 of the human Melatonin receptor 1A (MTNR1A) gene, is expressed in adult human tissues (testes and brain) and in the human cell line HEK 293. Although the role of piR_015520 expression in brain tissue remains unknown, the testes-specific expression is consistent with previous findings in several species. Surprisingly, in contrast to the mechanism known for miRNA-mediated modulation of gene expression, piRNA_015520 negatively regulates MTNR1A gene expression by binding to its genomic region. This finding suggests that changes in individual piRNA levels could influence both autoregulatory gene expression and the expression of the gene in which the piRNA is located. These findings offer a new perspective for piRNAs functioning as gene regulators in humans.     

Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus.

The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.     

福氏志贺痢疾杆菌硫胺素单磷酸激酶(ThiL)表达纯化及晶体生长研究

硫胺素单磷酸激酶(ThiL)在ATP存在下催化硫胺素单磷酸(TMP)形成硫胺素焦磷酸(TPP)和ADP,硫胺素焦磷酸就是维生素B1的活性形式。硫胺素单磷酸激酶属于一个小的ATP结合蛋白超家族成员。将来源于福氏志贺痢疾杆菌2a(301株)ThiL基因构建入pET-22b(+)表达载体,在大肠杆菌中得到高效表达,经过两步纯化,得到高纯度蛋白,用于晶体生长,经过对其晶体生长条件进行摸索和优化,得到了能用于X-射线衍射的单晶,为其结构解析、催化机理研究和药物设计提供了基础。