Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA
Crc is a global modulator involved in Carbon Catabolite Repression (CCR) in Pseudomonas, manipulating the uptake and assimilation of preferred carbon substrates. It is postulated that the free Crc is regulated by a complex and yet unknown process involving small non-coding regulatory RNAs, CrcY and...
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ftdoajarticles:oai:doaj.org/article:679c9cb5eb8a40ac84033c59d311b4c2 2023-05-15T13:52:13+02:00 Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA Rakhi Sharma Jaydeep Paul Sneha Paturi Malay K. Ray Mandar V. Deshmukh 2022-06-01T00:00:00Z https://doi.org/10.1016/j.jmro.2022.100047 https://doaj.org/article/679c9cb5eb8a40ac84033c59d311b4c2 EN eng Elsevier http://www.sciencedirect.com/science/article/pii/S2666441022000176 https://doaj.org/toc/2666-4410 2666-4410 doi:10.1016/j.jmro.2022.100047 https://doaj.org/article/679c9cb5eb8a40ac84033c59d311b4c2 Journal of Magnetic Resonance Open, Vol 10, Iss , Pp 100047- (2022) Crc CrcY and CrcZ Hfq Pseudomonas syringae Lz4W backbone dynamics and RNA binding protein Medical physics. Medical radiology. Nuclear medicine R895-920 Physics QC1-999 article 2022 ftdoajarticles https://doi.org/10.1016/j.jmro.2022.100047 2022-12-31T02:46:00Z Crc is a global modulator involved in Carbon Catabolite Repression (CCR) in Pseudomonas, manipulating the uptake and assimilation of preferred carbon substrates. It is postulated that the free Crc is regulated by a complex and yet unknown process involving small non-coding regulatory RNAs, CrcY and CrcZ. While Crc's interaction with AANAANAA rich target mRNA was proposed initially, but the conclusive proof was never obtained. Recent cryoEM structural studies derived a ternary complex formed by the hexameric RNA chaperon Hfq, Crc, and an mRNA fragment to highlight the assembly of these major elements in P. aeruginosa CCR. Although Crc was shown to kinetically favor and stabilize the association between Hfq with RNA, the interaction between Crc and substrate RNA remains enigmatic. Here, we present the solution structure, dynamics, and function of Crc (∼ 31 kDa, PsCrc) from a psychrotrophic Antarctic bacterium P. syringae Lz4W exclusively using NMR spectrometer operating at 600 MHz 1H frequency. Further, by utilizing several in vitro biophysical and in vivo functional assays to study the interaction between PsCrc and a representative small RNA as well as endogenous CrcY and CrcZ RNA, we demonstrate the significance of residues D100 and K101 in the activity of PsCrc. Our studies suggest that PsCrc has divergently evolved from Apurinic/apyrimidinic (AP) endonuclease by gaining a non-canonical RNA binding region. Our study proposes a dynamic and complex association of PsCrc:CrcY/Z RNA and Hfq:Crc:mRNA to regulate inactive and active CCR. Moreover, the combination of existing solution NMR approaches used in this study paves a path for meaningfully studying high MW proteins with the help of relatively lower field-strength spectrometers. Article in Journal/Newspaper Antarc* Antarctic Directory of Open Access Journals: DOAJ Articles Antarctic Journal of Magnetic Resonance Open 10-11 100047 |
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ftdoajarticles |
language |
English |
topic |
Crc CrcY and CrcZ Hfq Pseudomonas syringae Lz4W backbone dynamics and RNA binding protein Medical physics. Medical radiology. Nuclear medicine R895-920 Physics QC1-999 |
spellingShingle |
Crc CrcY and CrcZ Hfq Pseudomonas syringae Lz4W backbone dynamics and RNA binding protein Medical physics. Medical radiology. Nuclear medicine R895-920 Physics QC1-999 Rakhi Sharma Jaydeep Paul Sneha Paturi Malay K. Ray Mandar V. Deshmukh Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA |
topic_facet |
Crc CrcY and CrcZ Hfq Pseudomonas syringae Lz4W backbone dynamics and RNA binding protein Medical physics. Medical radiology. Nuclear medicine R895-920 Physics QC1-999 |
description |
Crc is a global modulator involved in Carbon Catabolite Repression (CCR) in Pseudomonas, manipulating the uptake and assimilation of preferred carbon substrates. It is postulated that the free Crc is regulated by a complex and yet unknown process involving small non-coding regulatory RNAs, CrcY and CrcZ. While Crc's interaction with AANAANAA rich target mRNA was proposed initially, but the conclusive proof was never obtained. Recent cryoEM structural studies derived a ternary complex formed by the hexameric RNA chaperon Hfq, Crc, and an mRNA fragment to highlight the assembly of these major elements in P. aeruginosa CCR. Although Crc was shown to kinetically favor and stabilize the association between Hfq with RNA, the interaction between Crc and substrate RNA remains enigmatic. Here, we present the solution structure, dynamics, and function of Crc (∼ 31 kDa, PsCrc) from a psychrotrophic Antarctic bacterium P. syringae Lz4W exclusively using NMR spectrometer operating at 600 MHz 1H frequency. Further, by utilizing several in vitro biophysical and in vivo functional assays to study the interaction between PsCrc and a representative small RNA as well as endogenous CrcY and CrcZ RNA, we demonstrate the significance of residues D100 and K101 in the activity of PsCrc. Our studies suggest that PsCrc has divergently evolved from Apurinic/apyrimidinic (AP) endonuclease by gaining a non-canonical RNA binding region. Our study proposes a dynamic and complex association of PsCrc:CrcY/Z RNA and Hfq:Crc:mRNA to regulate inactive and active CCR. Moreover, the combination of existing solution NMR approaches used in this study paves a path for meaningfully studying high MW proteins with the help of relatively lower field-strength spectrometers. |
format |
Article in Journal/Newspaper |
author |
Rakhi Sharma Jaydeep Paul Sneha Paturi Malay K. Ray Mandar V. Deshmukh |
author_facet |
Rakhi Sharma Jaydeep Paul Sneha Paturi Malay K. Ray Mandar V. Deshmukh |
author_sort |
Rakhi Sharma |
title |
Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA |
title_short |
Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA |
title_full |
Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA |
title_fullStr |
Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA |
title_full_unstemmed |
Crc of Pseudomonas syringae Lz4W utilizes the dominant RNA binding site for mutually exclusive interactions with Hfq:mRNA and CrcY/Z RNA |
title_sort |
crc of pseudomonas syringae lz4w utilizes the dominant rna binding site for mutually exclusive interactions with hfq:mrna and crcy/z rna |
publisher |
Elsevier |
publishDate |
2022 |
url |
https://doi.org/10.1016/j.jmro.2022.100047 https://doaj.org/article/679c9cb5eb8a40ac84033c59d311b4c2 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Journal of Magnetic Resonance Open, Vol 10, Iss , Pp 100047- (2022) |
op_relation |
http://www.sciencedirect.com/science/article/pii/S2666441022000176 https://doaj.org/toc/2666-4410 2666-4410 doi:10.1016/j.jmro.2022.100047 https://doaj.org/article/679c9cb5eb8a40ac84033c59d311b4c2 |
op_doi |
https://doi.org/10.1016/j.jmro.2022.100047 |
container_title |
Journal of Magnetic Resonance Open |
container_volume |
10-11 |
container_start_page |
100047 |
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1766256497936826368 |