Allosteric mechanism of the V. vulnificus adenine riboswitch resolved by four-dimensional chemical mapping

Abstract
Data availability
Article and author information
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Abstract

The structural interconversions that mediate the gene regulatory functions of RNA molecules may be different from classic models of allostery, but the relevant structural correlations have remained elusive in even intensively studied systems. Here, we present a four-dimensional expansion of chemical mapping called lock-mutate-map-rescue (LM²R), which integrates multiple layers of mutation with nucleotide-resolution chemical mapping. This technique resolves the core mechanism of the adenine-responsive V. vulnificus add riboswitch, a paradigmatic system for which both Monod-Wyman-Changeux (MWC) conformational selection models and non-MWC alternatives have been proposed. To discriminate amongst these models, we locked each functionally important helix through designed mutations and assessed formation or depletion of other helices via compensatory rescue evaluated by chemical mapping. These LM²R measurements give strong support to the pre-existing correlations predicted by MWC models, disfavor alternative models, and suggest additional structural heterogeneities that may be general across ligand-free riboswitches.

Data availability

The following data sets were generated

1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 WT no ligand
ADD140_1M7_0001.

https://rmdb.stanford.edu/detail/ADD140_1M7_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 WT w/ ligand
ADD140_1M7_0002.

https://rmdb.stanford.edu/detail/ADD140_1M7_0002
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 A109U no ligand
ADD140_1M7_0003.

https://rmdb.stanford.edu/detail/ADD140_1M7_0003
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 A109U w/ ligand
ADD140_1M7_0004.

https://rmdb.stanford.edu/detail/ADD140_1M7_0004
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 G78C no ligand
ADD140_1M7_0005.

https://rmdb.stanford.edu/detail/ADD140_1M7_0005
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 G78C w/ ligand
ADD140_1M7_0006.

https://rmdb.stanford.edu/detail/ADD140_1M7_0006
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 A116U no ligand
ADD140_1M7_0007.

https://rmdb.stanford.edu/detail/ADD140_1M7_0007
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 A116U w/ ligand
ADD140_1M7_0008.

https://rmdb.stanford.edu/detail/ADD140_1M7_0008
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 G44C no ligand
ADD140_1M7_0009.

https://rmdb.stanford.edu/detail/ADD140_1M7_0009
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 G44C w/ ligand
ADD140_1M7_0010.

https://rmdb.stanford.edu/detail/ADD140_1M7_0010
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 G37C no ligand
ADD140_1M7_0011.

https://rmdb.stanford.edu/detail/ADD140_1M7_0011
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 G37C w/ ligand
ADD140_1M7_0012.

https://rmdb.stanford.edu/detail/ADD140_1M7_0012
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 G81C no ligand
ADD140_1M7_0013.

https://rmdb.stanford.edu/detail/ADD140_1M7_0013
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2 G81C w/ ligand
ADD140_1M7_0014.

https://rmdb.stanford.edu/detail/ADD140_1M7_0014
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2R 1-bp no ligand
ADD140_RSQ_0001.

https://rmdb.stanford.edu/detail/ADD140_RSQ_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2R 1-bp w/ ligand
ADD140_RSQ_0002.

https://rmdb.stanford.edu/detail/ADD140_RSQ_0002
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2R 2-bp no ligand
ADD140_RSQ_0003.

https://rmdb.stanford.edu/detail/ADD140_RSQ_0003
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 M2R 2-bp w/ ligand
ADD140_RSQ_0004.

https://rmdb.stanford.edu/detail/ADD140_RSQ_0004
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 LM2R lock-P1 no ligand
ADD140_LCK_0001.

https://rmdb.stanford.edu/detail/ADD140_LCK_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 LM2R lock-P2 no ligand
ADD140_LCK_0002.

https://rmdb.stanford.edu/detail/ADD140_LCK_0002
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 LM2R lock-P4A no ligand
ADD140_LCK_0003.

https://rmdb.stanford.edu/detail/ADD140_LCK_0003
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 LM2R lock-P4B no ligand
ADD140_LCK_0004.

https://rmdb.stanford.edu/detail/ADD140_LCK_0004
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 LM2R lock-P1B no ligand
ADD140_LCK_0005.

https://rmdb.stanford.edu/detail/ADD140_LCK_0005
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 LM2R mut-P2 no ligand
ADD140_LCK_0006.

https://rmdb.stanford.edu/detail/ADD140_LCK_0006
1. Tian S
2. Kladwang W
3. Das R
(2017) add 71 1D standard states
ADD71_STD_0001.

https://rmdb.stanford.edu/detail/ADD71_STD_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) add 128 1D standard states
ADD128_STD_0001.

https://rmdb.stanford.edu/detail/ADD128_STD_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 1D standard states
ADD140_STD_0001.

https://rmdb.stanford.edu/detail/ADD140_STD_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 deep-chemical profiling no ligand
ADD140_DCP_0001.

https://rmdb.stanford.edu/detail/ADD140_DCP_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) add 140 deep-chemical profiling w/ ligand
ADD140_DCP_0002.

https://rmdb.stanford.edu/detail/ADD140_DCP_0002
1. Tian S
2. Kladwang W
3. Das R
(2017) GIR1 ribozyme M2R
RNAPZ5_RSQ_0001.

https://rmdb.stanford.edu/detail/RNAPZ5_RSQ_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) P4P6 domain M2R
TRP4P6_RSQ_0001.

https://rmdb.stanford.edu/detail/TRP4P6_RSQ_0001
1. Tian S
2. Kladwang W
3. Das R
(2017) 16S FWJ domain M2R
16SFWJ_RSQ_0001.

https://rmdb.stanford.edu/detail/16SFWJ_RSQ_0001

Article and author information

Author details

Siqi Tian

Department of Biochemistry, Stanford University, Stanford, United States

Competing interests
The authors declare that no competing interests exist.
Wipapat Kladwang

Department of Biochemistry, Stanford University, Stanford, United States

Competing interests
The authors declare that no competing interests exist.
Rhiju Das

Department of Biochemistry, Stanford University, Stanford, United States

For correspondence
rhiju@stanford.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7497-0972

Funding

National Institutes of Health (R01GM102519)

Rhiju Das

National Institutes of Health (R35GM122579)

Rhiju Das

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.