Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes

ABSTRACT Combinatorial or random methods for strain engineering have been extensively used for the improvement of multigenic phenotypes and other traits for which the underlying mechanism is not fully understood. Although the preferred method has traditionally been mutagenesis and selection, our laboratory has successfully used mutant transcription factors, which direct the RNA polymerase (RNAP) during transcription, to engineer complex phenotypes in microbial cells. Here, we show that it is also possible to impart new phenotypes by altering the RNAP core enzyme itself, in particular through mutagenesis of the alpha subunit of the bacterial polymerase. We present the use of this tool for improving tolerance of Escherichia coli to butanol and other solvents and for increasing the titers of two commercially relevant products, l-tyrosine and hyaluronic acid. In addition, we explore the underlying physiological changes that give rise to the solvent-tolerant mutant.

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The Phage-Encoded N-Acetyltransferase Rac Mediates Inactivation of Pseudomonas aeruginosa Transcription by Cleavage of the RNA Polymerase Alpha Subunit

Viruses ◽

10.3390/v12090976 ◽

2020 ◽

Vol 12 (9) ◽

pp. 976 ◽

Cited By ~ 1

Author(s):

Pieter-Jan Ceyssens ◽

Jeroen De Smet ◽

Jeroen Wagemans ◽

Natalia Akulenko ◽

Evgeny Klimuk ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Rna Polymerase ◽

Transcription Initiation ◽

Alpha Subunit ◽

Regulation Mechanism ◽

Post Translational Modification ◽

Α Subunit ◽

Bacterial Transcription ◽

Highly Active ◽

Bacterial Rna

In this study, we describe the biological function of the phage-encoded protein RNA polymerase alpha subunit cleavage protein (Rac), a predicted Gcn5-related acetyltransferase encoded by phiKMV-like viruses. These phages encode a single-subunit RNA polymerase for transcription of their late (structure- and lysis-associated) genes, whereas the bacterial RNA polymerase is used at the earlier stages of infection. Rac mediates the inactivation of bacterial transcription by introducing a specific cleavage in the α subunit of the bacterial RNA polymerase. This cleavage occurs within the flexible linker sequence and disconnects the C-terminal domain, required for transcription initiation from most highly active cellular promoters. To achieve this, Rac likely taps into a novel post-translational modification (PTM) mechanism within the host Pseudomonas aeruginosa. From an evolutionary perspective, this novel phage-encoded regulation mechanism confirms the importance of PTMs in the prokaryotic metabolism and represents a new way by which phages can hijack the bacterial host metabolism.

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Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase

Nucleic Acids Research ◽

10.1093/nar/gkz1221 ◽

2020 ◽

Vol 48 (4) ◽

pp. 2144-2155 ◽

Cited By ~ 3

Author(s):

Yeonoh Shin ◽

Mark Hedglin ◽

Katsuhiko S Murakami

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Initiation Complex ◽

X Ray ◽

The Core ◽

Core Enzyme ◽

Bacterial Rna ◽

Reiterative Transcription ◽

Transcript Initiation ◽

Template Dna

Abstract Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak–trigger–freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.

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Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.666504 ◽

2021 ◽

Vol 8 ◽

Author(s):

Virtu Solano-Collado ◽

Sofía Ruiz-Cruz ◽

Fabián Lorenzo-Díaz ◽

Radoslaw Pluta ◽

Manuel Espinosa ◽

...

Keyword(s):

Rna Polymerase ◽

Sigma Factor ◽

Regulation Of Gene Expression ◽

E Coli ◽

Core Enzyme ◽

Protein Architecture ◽

Bacterial Rna ◽

Promoter Recognition ◽

Rna Polymerase Holoenzyme

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

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The plastid rpoA gene encoding a protein homologous to the bacterial RNA polymerase alpha subunit is expressed in pea chloroplasts

MGG Molecular & General Genetics ◽

10.1007/bf00330945 ◽

1989 ◽

Vol 217 (1) ◽

pp. 77-84 ◽

Cited By ~ 31

Author(s):

Saul Purton ◽

John C. Gray

Keyword(s):

Rna Polymerase ◽

Alpha Subunit ◽

Gene Encoding ◽

Bacterial Rna ◽

Rpoa Gene

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Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase

10.1101/732214 ◽

2019 ◽

Author(s):

Yeonoh Shin ◽

Mark Hedglin ◽

Katsuhiko S. Murakami

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

X Ray ◽

The Core ◽

Core Enzyme ◽

Biochemical Assays ◽

Bacterial Rna ◽

Reiterative Transcription ◽

Template Dna ◽

Rna Transcript

ABSTRACTReiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains 8 poly-G RNA bases synthesized using 3 C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak trigger freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Structures and biochemical assays revealed how the RNA transcript from the pyrG promoter is guided toward the Rifampin-binding pocket then the main channel of RNA polymerase and provided insight into RNA slippage during reiterative transcription of the pyrG promoter. Lastly, we determined a structure of a reiterative transcription complex at the pyrBI promoter and revealed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.SIGNIFICANCE STATEMENTRNA polymerase synthesizes multiple bases of RNA using a single base of the template DNA due to slippage between RNA transcript and template DNA. This noncanonical RNA synthesis is called “reiterative transcription,” playing several regulatory roles cellular organisms and viruses. In this study, we determined a series of X-ray crystal structures of a bacterial RNA polymerase engaged in reiterative transcription and characterized a role of template DNA during reiterative transcription by biochemical assays. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. We also provide insights into the regulation of gene expression using two alternative ways of reiterative transcription.

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