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M B Elliott

Publications and source records attributed to M B Elliott.

5 recordsLinked to original sources
PubMed2001

The mechanism of RNA binding to TRAP: initiation and cooperative interactions.

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis is an 11-subunit protein that binds a series of 11 GAG and UAG repeats separated by two to three-spacer nucleosides in trp leader mRNA. The structure of TRAP bound to an RNA containing 11 GAG repeats shows that the RNA wraps around the outside of the protein ring with each GAG interacting with the protein in nearly identical fashion. The only direct hydrogen bond interactions between the protein and the RNA backbone are to the 2'-hydroxyl groups on the third G of each repeat. Replacing all 11 of these guanosines with deoxyriboguanosine eliminates measurable binding to TRAP. In contrast, a single riboguanosine in an otherwise entirely DNA oligonucleotide dramatically stabilizes TRAP binding, and facilitates the interaction of the remaining all-DNA portion with the protein. Studies of TRAP binding to RNAs with between 2 and 11 GAGs, UAGs, AAGs, or CAGs showed that the stability of a TRAP-RNA complex is not directly proportional to the number of repeats in the RNA. These studies also showed that the effect of the identity of the residue in the first position of the triplet, with regard to binding to TRAP, is dependent on the number of repeats in the RNA. Together these data support a model in which TRAP binds to RNA by first forming an initial complex with a small subset of the repeats followed by a cooperative interaction with the remaining triplets.

5' Untranslated Regions↗
PubMed1999

Probing the TRAP-RNA interaction with nucleoside analogs.

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 5'-(UAGCC)-3' repeated 11 times and measured their effect on the TRAP-RNA interaction. Deoxyribonucleoside substitutions revealed that a 2'-hydroxyl group (2'-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 2'-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding.

Bacillus subtilis↗
PubMed1998

RNA structure inhibits the TRAP (trp RNA-binding attenuation protein)-RNA interaction.

TRAP (trp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus subtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer nucleotides. Previous mutagenesis experiments have identified three TRAP residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA binding. The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer. In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)11) form stable structures that inhibit binding to TRAP. This conclusion is based on the effects of temperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers combined with UV hyperchromicity and circular dichroism. Furthermore, introducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)11 RNAs stabilized the TRAP-RNA interaction. This effect was associated with decreased stability of the RNA structure as measured by circular dichroism spectroscopy. The precise nature of the structure of the ((G/U)AGCC)11 RNAs is not known but evidence is presented that it involves noncanonical interactions. We also observed that substitution of Arg-58 with Lys further reduced the ability of TRAP to interact with structured RNAs. Since in vivo function of TRAP may involve binding to structured RNAs, we suggest a potential function for this residue, which is conserved in TRAP from three different bacilli.

Bacillus↗