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The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl‐binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in‐line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12‐RNA interactions. When introducing the same modification to AdoCbl the influence of any side‐chain modification tested is reduced. This renders the impact of the adenosyl‐ligand for B12‐btuB riboswitch recognition clearly beyond the known role in affinity. [ABSTRACT FROM AUTHOR]

Abstract: The organometallic complex coenzyme B12 (adenosyl cobalamin, AdoCbl) is not only an essential coenzyme in many biochemical reactions of most if not all living organisms but has lately been shown to play a crucial role in the regulation of B12 related genes. As a consequence, coenzyme B12 has been a target of intense research. However, the investigations of AdoCbl have often been hampered due to its high light-sensitivity leading to decomposition of the compound within a few seconds. Here, we describe a strategy to synthesize more light-stable coenzyme B12 analogs, which show similar steric properties as adenosyl cobalamin. The synthesis, structural characterization as well as the pH dependent “base-on/base-off” behavior of cyanide bridged vitamin B12 conjugates with either a cis-[(NH3)2Pt]2+ or an [enPt]2+ moiety, leading to cis-[(NH3)2PtCl-vitB12]+ (1) and [enPtCl-vitB12]+ (2) are reported. The subsequent reaction of cis-[(NH3)2PtCl-vitB12]+ with the model nucleobase 9-methyladenine leads to the corresponding adduct, where the adenine moiety is coordinated to the Pt2+ center either via N1 or N7. This compound is light-stable and harbors the adenine moiety in the same distance of 5Å above the corrin plane as present in the highly light-sensitive adenosyl cobalamin. [Copyright &y& Elsevier]

By applying four different acid derivatives of vitamin B12, we demonstrate that the H-bonding pattern and the electrostatic environment provided by each side chain of the corrin ring are crucial for the correct structural rearrangement of the btuBriboswitch of E. coli. [ABSTRACT FROM AUTHOR]

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B 12 transport protein BtuB by specifically binding coenzyme B 12 (AdoCbl) as its natural ligand. The B 12 sensing riboswitch class is known to accept various B 12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B 12 show larger effects indicating crucial B 12 -RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B 12 -btuB riboswitch recognition clearly beyond the known role in affinity., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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 Mg 2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg 2+ induced tertiary interactions of the btuB riboswitch from E. coli , we here report Mg 2+ 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, Tb 3+ being an established Mg 2+ mimic. Interestingly many of the M n+ ( n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B 12 -binding riboswitches. Comparison with the published crystal structure of the coenzyme B 12 riboswitch of S. thermophilum aided in identifying a common set of M n+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that M n+ 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 B 12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these M n+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb 3+ 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.

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.

By applying four different acid derivatives of vitamin B(12), we demonstrate that the H-bonding pattern and the electrostatic environment provided by each side chain of the corrin ring are crucial for the correct structural rearrangement of the btuB riboswitch of E. coli.