Coordination among tertiary base pairs results in an efficient frameshift-stimulating RNA pseudoknot.
Frameshifting is an essential process that regulates protein synthesis in many viruses. The ribosome may slip backward when encountering a frameshift motif on the messenger RNA, which usually contains a pseudoknot structure involving tertiary base pair interactions. Due to the lack of detailed molecular explanations, previous studies investigating which features of the pseudoknot are important to stimulate frameshifting have presented diverse conclusions. Here we constructed a bimolecular pseudoknot to dissect the interior tertiary base pairs and used single-molecule approaches to assess the structure targeted by ribosomes. We found that the first ribosome target stem was resistant to unwinding when the neighboring loop was confined along the stem; such constrained conformation was dependent on the presence of consecutive adenosines in this loop. Mutations that disrupted the distal base triples abolished all remaining tertiary base pairs. Changes in frameshifting efficiency correlated with the stem unwinding resistance. Our results demonstrate that various tertiary base pairs are coordinated inside a highly efficient frameshift-stimulating RNA pseudoknot and suggest a mechanism by which mechanical resistance of the pseudoknot may persistently act on translocating ribosomes.
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.
Yu-Ting
YT
Chen
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.
Kai-Chun
KC
Chang
Institute of Biochemistry, National Chung-Hsing University, Taichung 40227, Taiwan.
Hao-Teng
HT
Hu
Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei 10617, Taiwan.
Yi-Lan
YL
Chen
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.
You-Hsin
YH
Lin
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.
Chiung-Fang
CF
Hsu
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.
Cheng-Fu
CF
Chang
Institute of Biochemistry, National Chung-Hsing University, Taichung 40227, Taiwan.
Kung-Yao
KY
Chang
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.|Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei 10617, Taiwan.|Department of Life Science, National Taiwan University, Taipei 10617, Taiwan.
Jin-Der
JD
Wen
D020029:Base Pairing; D031541:Fluorescence Resonance Energy Transfer; D018965:Frameshifting, Ribosomal / Q000502:physiology*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D052898:Optical Tweezers; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D016364:Reading Frames; D012270:Ribosomes / Q000378:metabolism*; D013379:Substrate Specificity
D009843:Oligoribonucleotides; D012333:RNA, Messenger