Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Qiguo Yu; Tarinee Tungsuchat-Huang; Alexander Ioannou; Alice Barkan; Pal Maliga

doi:10.1111/tpj.16930

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Qiguo Yu, Tarinee Tungsuchat-Huang, Alexander Ioannou, Alice Barkan, Pal Maliga

Waksman Institute of Microbiology

Rutgers, The State University

Research output: Contribution to journal › Article › peer-review

Abstract

Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. In this study, we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5′ UTR, which harbors a binding site for PPR10, a protein that activates atpH at the posttranscriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5′ UTRs in different design contexts. Notably, high GFP expression was not coupled to the stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), presumably by promoting such rapid RNA cleavage and 5′ exonucleolytic degradation that PPR10 had insufficient time to bind and protect gfp RNA, resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5′ UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning of reporter gene expression across a wide range, spanning from a mere 0.02–25% of the total soluble cellular protein. These findings highlight the potential of employing cis-elements from heterologous species and expand the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.

Original language	American English
Pages (from-to)	2437-2449
Number of pages	13
Journal	Plant Journal
Volume	119
Issue number	5
DOIs	https://doi.org/10.1111/tpj.16930
State	Published - Sep 2024

ASJC Scopus subject areas

Genetics
Plant Science
Cell Biology

Keywords

Nicotiana tabacum (tobacco)
PPR10 RNA binding protein
chloroplast gene expression
posttranscriptional regulation
synthetic operons
tRNA cis-element

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10.1111/tpj.16930

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title = "Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons",

abstract = "Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. In this study, we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5′ UTR, which harbors a binding site for PPR10, a protein that activates atpH at the posttranscriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5′ UTRs in different design contexts. Notably, high GFP expression was not coupled to the stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), presumably by promoting such rapid RNA cleavage and 5′ exonucleolytic degradation that PPR10 had insufficient time to bind and protect gfp RNA, resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5′ UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning of reporter gene expression across a wide range, spanning from a mere 0.02–25% of the total soluble cellular protein. These findings highlight the potential of employing cis-elements from heterologous species and expand the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.",

keywords = "Nicotiana tabacum (tobacco), PPR10 RNA binding protein, chloroplast gene expression, posttranscriptional regulation, synthetic operons, tRNA cis-element",

author = "Qiguo Yu and Tarinee Tungsuchat-Huang and Alexander Ioannou and Alice Barkan and Pal Maliga",

note = "Publisher Copyright: {\textcopyright} 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.",

year = "2024",

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doi = "10.1111/tpj.16930",

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T1 - Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

AU - Yu, Qiguo

AU - Tungsuchat-Huang, Tarinee

AU - Ioannou, Alexander

AU - Barkan, Alice

AU - Maliga, Pal

PY - 2024/9

Y1 - 2024/9

N2 - Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. In this study, we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5′ UTR, which harbors a binding site for PPR10, a protein that activates atpH at the posttranscriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5′ UTRs in different design contexts. Notably, high GFP expression was not coupled to the stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), presumably by promoting such rapid RNA cleavage and 5′ exonucleolytic degradation that PPR10 had insufficient time to bind and protect gfp RNA, resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5′ UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning of reporter gene expression across a wide range, spanning from a mere 0.02–25% of the total soluble cellular protein. These findings highlight the potential of employing cis-elements from heterologous species and expand the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.

AB - Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. In this study, we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5′ UTR, which harbors a binding site for PPR10, a protein that activates atpH at the posttranscriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5′ UTRs in different design contexts. Notably, high GFP expression was not coupled to the stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), presumably by promoting such rapid RNA cleavage and 5′ exonucleolytic degradation that PPR10 had insufficient time to bind and protect gfp RNA, resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5′ UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning of reporter gene expression across a wide range, spanning from a mere 0.02–25% of the total soluble cellular protein. These findings highlight the potential of employing cis-elements from heterologous species and expand the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.

KW - Nicotiana tabacum (tobacco)

KW - PPR10 RNA binding protein

KW - chloroplast gene expression

KW - posttranscriptional regulation

KW - synthetic operons

KW - tRNA cis-element

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JO - Plant Journal

JF - Plant Journal

IS - 5

ER -

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

Abstract

ASJC Scopus subject areas

Keywords

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