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.
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260, USA.
S
S
Xirasagar
M B
MB
Elliott
W
W
Bartolini
P
P
Gollnick
P A
PA
Gottlieb
D001407:Bacillus / Q000235:genetics; D001426:Bacterial Proteins; D001665:Binding Sites; D002942:Circular Dichroism; D017124:Conserved Sequence; D006151:Guanosine / Q000031:analogs & derivatives; D009154:Mutation; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012641:Selection, Genetic; D013056:Spectrophotometry, Ultraviolet; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D006151:Guanosine; C068218:7-deazaguanosine; D012313:RNA