The ω subunit of the RNA polymerase and the Ssr1600 protein play roles in high CO2 acclimation of the cyanobacterium Synechocystis sp. PCC 6803
Ezu, Shedrack (2020-01-05)
The ω subunit of the RNA polymerase and the Ssr1600 protein play roles in high CO2 acclimation of the cyanobacterium Synechocystis sp. PCC 6803
(05.01.2020)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
suljettu
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe202001283728
https://urn.fi/URN:NBN:fi-fe202001283728
Tiivistelmä
The transcription machinery of cyanobacteria includes the RNA polymerase (RNAP) with core composition of α2ββ’γω. For transcription initiation, the RNAP core recruits one of the σ factors. The ω subunit of RNAP is non-essential when cells are grown in ambient air, but essential in high CO2. However, the phenotype of the ω-disrupted strain (ΔrpoZ) is intermittently reverted back to high CO2 competent. In two separately isolated revertant strains, a unique secondary mutation was found from the ssr1600 gene (L24S in ∆rpoZ-R1 and G53V in ∆rpoZ-R2). The Ssr1600 protein is a homolog of SpoIIAA identified in B. subtilis as an anti-σ-factor-antagonist.
The aims of this thesis are 1) to investigate if ssr1600 is in the same operon with the two adjacent genes slr0954 and slr0955, 2) to study if the ssr1600 operon responds to CO2 concentration, 3) to find out if the absence of the ω sub-unit or mutations in ssr1600 affects the recruitment of σ factors in different CO2 concentrations.
For operon analysis, DNA-free RNA was isolated, copied to cDNA and then tested with PCR if adjacent genes were in the same cDNA molecule or not. The wild type, ∆rpoZ, ∆rpoZ-R1 and ∆rpoZ-R2 were grown in ambient air and in 3% CO2, and transcript abundances of ssr1600 and slr0954 were detected with qPCR. To identify the amount of different σ factors in the RNAP holoenzyme, RNAP complexes were collected from strains with His-tag in the RNAP and western blot was performed.
Results showed that the ssr1600 gene forms an operon with slr0954 but not with slr0955. Similar amounts of ssr1600 and slr0954 transcripts were detected in all strains in ambient air and in high CO2 indicating that the ssr1600 operon is not regulated by CO2 concentration and not dependent on the ω-subunit of RNAP. The analysis of RNAP complexes revealed that the disruption of the ω subunit and the mutations in the Ssr1600 protein affects σ factor recruitment
The aims of this thesis are 1) to investigate if ssr1600 is in the same operon with the two adjacent genes slr0954 and slr0955, 2) to study if the ssr1600 operon responds to CO2 concentration, 3) to find out if the absence of the ω sub-unit or mutations in ssr1600 affects the recruitment of σ factors in different CO2 concentrations.
For operon analysis, DNA-free RNA was isolated, copied to cDNA and then tested with PCR if adjacent genes were in the same cDNA molecule or not. The wild type, ∆rpoZ, ∆rpoZ-R1 and ∆rpoZ-R2 were grown in ambient air and in 3% CO2, and transcript abundances of ssr1600 and slr0954 were detected with qPCR. To identify the amount of different σ factors in the RNAP holoenzyme, RNAP complexes were collected from strains with His-tag in the RNAP and western blot was performed.
Results showed that the ssr1600 gene forms an operon with slr0954 but not with slr0955. Similar amounts of ssr1600 and slr0954 transcripts were detected in all strains in ambient air and in high CO2 indicating that the ssr1600 operon is not regulated by CO2 concentration and not dependent on the ω-subunit of RNAP. The analysis of RNAP complexes revealed that the disruption of the ω subunit and the mutations in the Ssr1600 protein affects σ factor recruitment