TY  - JOUR
A1  - Ulbricht, Andrea
A1  - Nickel, Lisa
A1  - Weidenbach, Katrin
A1  - Vargas Gebauer, Herman
A1  - Kießling, Claudia
A1  - Förstner, Konrad U.
A1  - Schmitz, Ruth A.
T1  - The CARF protein MM_0565 affects transcription of the casposon-encoded cas1-solo gene in Methanosarcina mazei Gö1
JF  - Biomolecules
N2  - Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the cas genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which many have no reported function in CRISPR-mediated immunity. Here, we characterize MM_0565 associated to the type I-B CRISPR-locus of Methanosarcina mazei Gö1. We show that purified MM_0565 composed of a CRISPR-Cas Associated Rossmann Fold (CARF) and a winged helix-turn-helix domain forms a dimer in solution; in vivo, the dimeric MM_0565 is strongly stabilized under high salt stress. While direct effects on CRISPR-Cas transcription were not detected by genetic approaches, specific binding of MM_0565 to the leader region of both CRISPR-Cas systems was observed by microscale thermophoresis and electromobility shift assays. Moreover, overexpression of MM_0565 strongly induced transcription of the cas1-solo gene located in the recently reported casposon, the gene product of which shows high similarity to classical Cas1 proteins. Based on our findings, and taking the absence of the expressed CRISPR locus-encoded Cas1 protein into account, we hypothesize that MM_0565 might modulate the activity of the CRISPR systems on different levels.
KW  - methanoarchaea
KW  - CRISPR-Cas system
KW  - transcriptional regulation
KW  - adaptation phase
KW  - casposon
KW  - Methanosarcina mazei
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-211097
SN  - 2218-273X
VL  - 10
IS  - 8
ER  - 
TY  - JOUR
A1  - Jäger, Dominik
A1  - Pernitzsch, Sandy R.
A1  - Richter, Andreas S.
A1  - Backofen, Rolf
A1  - Sharma, Cynthia M.
A1  - Schmitz, Ruth A.
T1  - An archaeal sRNA targeting cis- and trans-encoded mRNAs via two distinct domains
JF  - Nucleic Acids Research
N2  - We report on the characterization and target analysis of the small (s) RNA\(_{162}\) in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5' fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA\(_{162}\) (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA\(_{162}\) is crucial for target interactions. Furthermore, our results indicate that sRNA\(_{162}\)-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 50 end of sRNA\(_{162}\) targets the 5'-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA\(_{162}\) acts as an antisense (as) RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated.
KW  - strain
KW  - escherichia coli
KW  - methanosarcina mazei GO1
KW  - methanol methyltransferase isozymes
KW  - small nucleolar RNAs
KW  - acetivorans C2A
KW  - antisense RNAs
KW  - GO1
KW  - transcriptional regulator
KW  - translational initiation
KW  - pyrococcus furiosus
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134972
VL  - 40
IS  - 21
ER  -