Tb3+-Cleavage Assays Reveal Specific Mg2+ Binding Sites Necessary to Pre-fold the btuB Riboswitch for AdoCbl Binding

Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement

Studies on Mg2+ and Coenzyme B12 Induced Conformational Changes in Coenzyme B12 Riboswitches from E. coli and D. hafniense

Coenzyme B12 riboswitches are widely distributed in prokaryotes and belong to one of the first reported metabolite binding RNA elements. This work focuses on coenzyme B12 riboswitches to understand the pre-organization of their highly conserved aptamer and high specificity towards coenzyme B12. To understand the aptamer architecture, we carried out in-line probing and terbium(III) cleavage experiments on the coenzyme B12 responsive btuB riboswitch from E. coli. We observed that Mg2+ plays a vital role in the pre-folding of the btuB riboswitch to facilitate its interaction with coenzyme B12. Moreover, the presence of Mg2+ binding sites near the conserved bases of the aptamer indicates the role of Mg2+ mediated tertiary interactions in aptamer pre-organization. We also studied the kinetics of interaction between the btuB riboswitch with its ligands using surface plasmon resonance spectroscopy. Our results indicate that the btuB aptamer has similar rates of association but varying rates of dissociation towards its ligands, justifying its different affinities towards its ligands. To explore further the diversity of B12 riboswitches, we characterized three B12 riboswitches from Desulfitobacterium hafniense. Our studies suggest that coenzyme B12 riboswitches could harbour variable structural elements to alter their affinity towards coenzyme B12 and regulate cobalamine metabolism within the cell. Together, our work highlights the significance of the aptamer architecture and ligand specificity for the functions of coenzyme B12 riboswitches in prokaryotes. Coenzym B12 Riboswitches sind in Prokaryoten weit verbreitet und waren unter den ersten bekannten Metabolit-bindenden RNA-Elementen. Die vorliegende Arbeit beschäftigt sich mit Coenzym B12 Riboswitches um ein besseres Verständnis für die Prä-Organisation des hochkonservierten Aptamers und die hohe Spezifität für Coenzym B12 zu erlangen. Zum besseren Verständnis der Aptamer-Architektur, wurden In-line Probing und Terbium(III)-Spaltungs-Experimente am Coenzym B12-sensitiven btuB riboswitch aus E.coli durchgeführt. Wir beobachten, dass Mg2+ entscheidend ist für eine Vorfaltung des btuB Riboswitches, welche die Wechselwirkung mit Coenzym B12 ermöglicht. Zudem weisen Mg2+-Bindungsstellen in der Nähe der konservierten Basen des Aptamers auf ein Mitwirken Mg2+-vermittelter tertiärer Wechselwirkungen bei der Prä-Organisation des Aptamers hin. Mittels Plasmon-Surface-Resonance-Spektroskopie haben wir auch die Kinetik der Wechselwirkung zwischen dem btuB Riboswitch und seinen Liganden untersucht. Unsere Ergebnisse zeigen ähnliche Assoziierungsraten verschiedener Liganden gegenüber dem btuB- Aptamer und es sind die Dissoziierungsraten, welche Unterschiede in den Affinitäten erklären. Um die Diversität von B12 Riboswitches weiter zu erkunden, haben wir drei Vertreter aus Desulfitobacterium hafniense untersucht. Unsere Ergebnisse weisen daraufhin, dass Coenzym B12 Riboswitches variable Strukturelemente beherbergen, welche die Affinität gegen Coenzym B12 beeinflussen und den Cobalamin-Stoffwechsel in der Zelle regulieren. Zusammengenommen zeigt unsere Arbeit die große Bedeutung von Aptamerarchitektur und Ligandenspezifität für die Funktionen der Coenzym B12 Riboswitches in Prokaryoten auf

Mg(2+)-induced conformational changes in the btuB riboswitch from E. coli

Mg2+ has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B12 (adenosyl-cobalamine, AdoCbl), and down-regulates the expression of the B12 transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg2+ and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg2+. With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg2+ (or low Mg2+ concentration) and the other appearing above ∼0.5 mM Mg2+. Distinct regions of the riboswitch exhibit different dissociation constants toward Mg2+, indicating a stepwise folding of the btuB RNA. Increasing the Mg2+ concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg2+ defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B12, and switch the RNA conformation. Moreover, raising the Mg2+ concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg2+ indicates a crucial role played by Mg2+ for defining an active conformation of the riboswitch

Monitoring Global Structural Changes and Specific Metal-Ion-Binding Sites in RNA by In-line Probing and Tb(III) Cleavage

In this chapter we describe the use of two methods, in-line probing as well as terbium(III) cleavage. Both methods can be applied to RNAs of any size, structure, and function. Aside from revealing directly metal ion-binding sites these techniques also provide structural information for longer RNA sequences that are out of range to be analyzed with other techniques such as NMR. The cleavage pattern derived from in-line probing experiments reflects local and overall conformational changes in RNA upon the addition of metal ions, metal complexes, or other ligands. On the other side, terbium(III) cleavage experiments are applied to locate specific metal ion-binding sites in RNA molecules

The AdoCbl–Riboswitch Interaction Investigated by In-Line Probing and Surface Plasmon Resonance Spectroscopy (SPR)

The unique feature of riboswitches is to control selected gene expression by specific recognition of a cognate ligand. AdoCbl (adenosyl cobalamin, coenzyme B12) riboswitches regulate the expression of enzymes and transporters involved in bacterial AdoCbl biosynthesis or uptake on a transcriptional and/or translational level. The analysis of ligand recognition and the induced conformational changes requires a detailed knowledge of the kinetics and thermodynamics of ligand binding and of the secondary structure rearrangements. This chapter describes the investigation of coenzyme B12 binding to the btuB riboswitch from Escherichia coli by in-line probing assays and surface plasmon resonance spectroscopy. The experimental conditions, requirements, and performance of both methods are presented together with the evaluation of the experimental data to determine the associated conformational changes of the RNA and the kinetic and thermodynamic parameters of ligand binding. Owing to the light sensitivity of the cobalt(I)′single bondcarbon bond, these methods were specifically modified to ensure the chemical integrity of AdoCbl

The corrinoid metabolism in desulfitobacterium hafniense - genetic and regulatory aspects

Corrinoids (e.g. vitamin B12, cobalamin) represent a family of complex organometallic cofactors essential to the metabolism of microorganisms, animal and humans, and are involved in several enzymes such as isomerases, methyltransferases and reductive dehalogenases. Microorganisms - and not all of them - however, are the only natural source of corrinoids. Desulfitobacterium hafniense is a strictly anaerobic bacterium harboring a versatile energy metabolism, including a process called organohalide respiration (OHR) during which chlorinated organic pollutants are used as terminal electron acceptors. The first genome sequences of D. hafniense isolates revealed the presence of a complete set of corrinoid biosynthesis genes together with many corrinoid-dependent pathways, such as OHR, and enzymes. We successfully cultivated D. hafniense strain TCE1 in absence of corrinoid in the medium, and preliminary HPLC analysis of extracted corrinoids suggested that strain TCE1 produces several types of corrinoids. A thorough analysis of corrinoid-related genes in the genomes of D. hafniense strains Y51 and DCB-2 revealed the presence of sixteen and eleven cobalamin riboswitches (Cbl-RS), respectively. Cbl-RS structures are known to regulate the transcription or translation of downstream located genes by corrinoid-dependent conformational changes occurring in the 5’-untranslated region of the corresponding RNA transcripts. In vitro in-line probing analysis of three significantly different riboswitches of D. hafniense highlighted their role as regulatory elements in the corrinoid metabolism. In vivo analysis of the transcription of the directly downstream genes upon addition of various corrinoids clearly confirmed it. Our study extends the knowledge on the functional diversity of cobalamin riboswitches

Regulation of the Corrinoid Metabolism in Desulfitobacteria

The corrinoid cofactor is essential to organohalide respiration (OHR) as key element in virtually all reductive dehalogenases. Desulfitobacteria, and more particularly D. hafniense, represent a group of versatile OHR bacteria able to produce corrinoids de novo, as initially discovered in the genome of D. hafniense Y51 (1). Since then several desulfitobacteria genomes have been sequenced revealing that the corrinoid metabolism (biosynthesis, transport, regulation) represents a conserved trait in a highly variable genetic background in the members of this genus. A thorough analysis of corrinoid-related genes in the genomes of D. hafniense strains Y51, DCB-2 (2) and TCE1 (3) revealed the presence of eighteen distinct cobalamin riboswitches (Cbl-RS). Cbl-RS structures are known to regulate the transcription or translation of downstream located genes by corrinoid-dependent conformational changes occurring in the 5’-untranslated region of the corresponding RNA transcripts. In vitro in-line probing analysis of three significantly different riboswitches of D. hafniense TCE1 highlighted their role as regulatory elements in the corrinoid metabolism. In vivo analysis of the transcription of the directly downstream genes upon addition of various corrinoids clearly confirmed it (4). Furthermore, we successfully cultivated D. hafniense TCE1 in the complete absence of corrinoid in the medium, and preliminary HPLC analysis suggested that strain TCE1 produces several types of corrinoids. The corrinoid metabolism of desulfitobacteria have made them excellent candidates for acquiring OHR metabolism (5), while their wide distribution in OHR ecological niches suggests that this genus might be an important source of corrinoids for members of the genera Dehalococcoides and Dehalobacter. (1) Nonaka et al. (2006), J Bacteriol 188:2262. (2) Kim et al. (2012), BMC Microbiol 8:12. (3) JGI Project ID 403245, coordinated by H. Smidt (Wageningen University). (4) Choudhary et al. (2013), J Bacteriol 195:5186. (5) Duret et al. (2012), Appl Environ Microbiol 78:6121

Estimation of tuberculosis incidence at subnational level using three methods to monitor progress towards ending TB in India, 2015–2020

Objectives We verified subnational (state/union territory (UT)/district) claims of achievements in reducing tuberculosis (TB) incidence in 2020 compared with 2015, in India.Design A community-based survey, analysis of programme data and anti-TB drug sales and utilisation data.Setting National TB Elimination Program and private TB treatment settings in 73 districts that had filed a claim to the Central TB Division of India for progress towards TB-free status.Participants Each district was divided into survey units (SU) and one village/ward was randomly selected from each SU. All household members in the selected village were interviewed. Sputum from participants with a history of anti-TB therapy (ATT), those currently experiencing chest symptoms or on ATT were tested using Xpert/Rif/TrueNat. The survey continued until 30 Mycobacterium tuberculosis cases were identified in a district.Outcome measures We calculated a direct estimate of TB incidence based on incident cases identified in the survey. We calculated an under-reporting factor by matching these cases within the TB notification system. The TB notification adjusted for this factor was the estimate by the indirect method. We also calculated TB incidence from drug sale data in the private sector and drug utilisation data in the public sector. We compared the three estimates of TB incidence in 2020 with TB incidence in 2015.Results The estimated direct incidence ranged from 19 (Purba Medinipur, West Bengal) to 1457 (Jaintia Hills, Meghalaya) per 100 000 population. Indirect estimates of incidence ranged between 19 (Diu, Dadra and Nagar Haveli) and 788 (Dumka, Jharkhand) per 100 000 population. The incidence using drug sale data ranged from 19 per 100 000 population in Diu, Dadra and Nagar Haveli to 651 per 100 000 population in Centenary, Maharashtra.Conclusion TB incidence in 1 state, 2 UTs and 35 districts had declined by at least 20% since 2015. Two districts in India were declared TB free in 2020