A search for the in trans role of GraL, an Escherichia coli small RNA
Maciej Dylewski , Monika Ćwiklińska , Katarzyna Potrykus
AbstractSmall RNA are very important post-transcriptional regu - lators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL’s targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one target’s activity ( nudE ) decreased under those conditions and was thus selected for further analysis. In the absence of GraL and greA , the nudE::lacZ fusion’s β-galactosidase activity was increased. However, a similar effect was also visible in the strain deleted only for greA . Furthermore, overpro - duction of GreA alone increased the nudE::lacZ fusion’s activity as well. This suggests existence of complex reg - ulatory loop-like interactions between GreA, GraL and nudE mRNA. To further dissect this relationship, we per - formed in vitro EMSA experiments employing GraL and nudE mRNA. However, stable GraL- nudE complexes were not detected, even though the detectable amount of un - bound GraL decreased as increasing amounts of nudE mRNA were added. Interestingly, GraL is being bound by Hfq, but nudE easily displaces it. We also conducted a search for genes that are synthetic lethal when deleted along with GraL. This revealed 40 genes that are ren - dered essential by GraL deletion, however, they are in - volved in many different cellular processes and no clear correlation was found. The obtained data suggest that GraL’s mechanism of action is non-canonical, unique and requires further research.
|Journal series||Acta Biochimica Polonica, ISSN 0001-527X, e-ISSN 1734-154X|
|Publication size in sheets||0.5|
|Keywords in English||GraL, GreA, sRNA, sRNA targets, synthetic lethal genes|
|License||Journal (articles only); published final; ; with publication|
|Score|| = 15.0, 26-04-2018, ArticleFromJournal|
= 15.0, 26-04-2018, ArticleFromJournal
|Publication indicators||: 2016 = 1.159 (2) - 2016=1.491 (5)|
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