Consensus sequence and secondary structure model of the most common form of Moco RNA motif derived from 176 representatives. R represents A or G and Y represents C or U. Boxed nucleotides denoted RBS are predicted to be the ribosome binding site for the adjacent ORF in some Moco RNA representatives.

A. Schematic representation of the moaABCDE operon of E. coli. Nucleotide numbers for the genome region upstream of the moaA ORF are established by defining the first transcription start site (S1) as +1 and a second transcription start site (S2) as −87. The approximate locations of the FNR and ModE binding sites are indicated with filled boxes, and the region designated as the Moco RNA motif begins and ends with the terminal nucleotides of P1 (Fig. 1). Numbering system and locations of various features are as reported previously (Anderson et al., 2000). B. Pathway for molybdenum cofactor biosynthesis in eubacteria (Schwarz, 2005). Proteins with abbreviated designations are enzymes in the pathway except for ModABC, which is an ABC-type molybdate transporter. The precise function of MoaB is unknown, but structural studies (Bader et al., 2004; Sanishvili et al., 2004) indicate that it is similar to MogA and therefore is likely to be involved in molybdopterin biosynthesis. Question marks indicate that the biosynthetic enzymes for the conversion are not known. Other proteins listed are enzymes that use Moco derivatives as coenzymes. Proteins whose coding regions are located downstream and near a Moco RNA motif in at least one organism are highlighted with black shading.

In-line probing assay of a Moco RNA. A. Sequence of the 138 moaA RNA of E. coli depicted to conform to the secondary structure model predicted from comparative sequence analysis data (Fig. 1A). Nucleotides shaded in grey undergo greater rates of spontaneous 3′ phosphodiester cleavage (from data depicted in B), which typically indicates an elevated level of structural flexibility relative to internucleotide linkages present in stable secondary or tertiary structures. The bar identifies purine nucleotides predicted to serve as a ribosome binding site and the translation start codon for moaA is boxed. B. Gel image of the products generated during an in-line probing assay of the 138 moaA RNA from E. coli. Lanes 1–3 contain the 5′³²P-labelled precursor RNA (Pre) loaded on the gel either without incubation (no reaction, NR), partially digested by T1 RNase (T1), or subjected to partial alkaline digestion (^–OH). Lane 4 was loaded with labelled precursor RNA after incubation under in-line probing conditions in the absence (–) of a possible ligand compound. Indicated are regions of the gel containing radiolabelled RNA fragments cleaved after nucleotides predicted to be base paired.

Moco RNA from E. coli requires Moco biosynthesis for gene repression. A. Depiction of the P3 region of the E. coli 149 moaA RNA whose corresponding DNA template was fused to a β-galactosidase reporter gene. M1 and M2 carry nucleotide changes as indicated relative to the wild-type (WT) RNA. B. Expression of β-galactosidase reporter mRNAs fused to the WT and mutant Moco RNAs under different Moco concentrations. Moco concentrations are elevated or reduced by the presence or absence of arabinose in the growth medium, respectively, due to the presence of a transgenic moa operon controlled by an arabinose-inducible promoter. C. Dependence of expression of the reporter genes described above on the availability of molybdate or tungstate in cells. The presence (modC) or absence (ΔmodC) of functional molybdate transporter protein in the E. coli strain used for the analysis is indicated. Cells were grown in LB containing 0.2% arabinose. Reporter gene expression assays were performed in triplicate (see Experimental procedures) and their mean values were plotted, with error bars indicating standard deviation for the replicates.

The effects of knockouts of various genes in the E. coli Moco biosynthetic pathway on expression of a β-galactosidase reporter gene fused to a DNA template for the WT 149 moaA RNA. Each transgenic strain carries a plasmid encoding E. coli moaABCDE under the control of an arabinose-inducible promoter.

The Moco RNA size and structure is similar to other riboswitch aptamers from E. coli. A. Comparison of the length of the E. coli moaA Moco aptamer with the lengths of regulatory RNA elements from E. coli that either are known to be bound by proteins or function as riboswitch aptamers. RNA targets for ribosomal proteins are not included in this analysis. B. Secondary structure models for the RNAs depicted in (A). RNAs not shown do not have secondary structures established. See the text for references.