Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy.
The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.
Schüttpelz
Mark
M
Applied Laser Physics and Laser Spectroscopy, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany. schuettp@physik.uni-bielefeld.de
Schöning
Jan C
JC
Doose
Sören
S
Neuweiler
Hannes
H
Peters
Elisabeth
E
Staiger
Dorothee
D
Sauer
Markus
M
D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D029681:Arabidopsis Proteins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C118496:GRP7 protein, Arabidopsis