Probing the TRAP–RNA interaction with nucleoside analogs

Abstract

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2-3 nonconserved spacer nucleotides in trp leader mRNA. To identify chemical groups on the RNA required for stability of the TRAP-RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 59-(UAGCC)-39 repeated 11 times and measured their effect on the TRAP-RNA interaction. Deoxyribonucleoside substitutions revealed that a 29-hydroxyl group (29-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP. The remaining hydroxyl groups are dispensable. Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP. In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability. Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP-RNA complex is provided by the AG dinucleotide of the triplet repeats. In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 29-OHs of (UAGCC) 11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding.

Thermodynamics of Tryptophan-Mediated Activation of the trp RNA-Binding Attenuation Protein

“…The tryptophan ligand is completely buried between the beta-sheets at the interface between subunits in a cavity that is largely apolar with a few polar side-chain and backbone moieties making hydrogen bond contacts to the sidechain amide and backbone amino and carboxyl groups of the tryptophan (12,14-16). The bound RNA wraps around the protein with conserved bases making specific contacts with TRAP residues (13,(16)(17)(18)(19).Until recently, limited data were available on the structure of inactivate, apo-TRAP; therefore, little was known about the allosteric mechanism of tryptophan activation of TRAP for RNAbinding. The oligomeric state of TRAP (11-mer) is not altered by tryptophan, remaining oligomeric over a wide range of concentrations (10 -6 -10 -2 M), ruling out assembly as an activation mechanism (20)(21)(22).…”

Posttranscription Initiation Control of Tryptophan Metabolism in Bacillus subtilis by the trp RNA-Binding Attenuation Protein (TRAP), anti-TRAP, and RNA Structure

“…The only direct hydrogen bond to the phosphodiester backbone is a hydrogen bond to the 2Ј OH of the ribose of the third G in each repeat. This contact, which was predicted by deoxyribonucleoside substitution studies (17), explains how TRAP distinguishes RNA from DNA. As predicted, Lys37, Lys56, and Arg58 all form hydrogen bonds with the RNA.…”

“…Nucleoside analog studies identified several important functional groups for interaction with TRAP on the second A and third G of each triplet repeat. These studies also suggested that the bases in the first position (G or U), as well as the spacer bases, are not crucial for TRAP recognition and binding (17).…”

Molecular basis of TRAP–5′SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism

“…The interaction between these triplets and TRAP has been well-characterized. The crystal structure of Bacillus stearothermophilus TRAP complexed with a synthetic RNA target indicates that Lys37, Lys56, and Arg58 (KKR) form hydrogen bonds with the A and the G residues in the second and third positions of the triplet repeat (Antson et al 1999;Elliott et al 1999). Gly18 and Asp39 form additional hydrogen bonds with the triplet repeats, and Phe32 likely participates in a stacking interaction (Hopcroft et al 2004).…”