Poster abstracts
Poster number 17 submitted by Kathryn Bormes
Role of Upstream RNA Secondary Structures in an RNA Thermometer from Bradyrhizobium japonicum
Kathryn M. Bormes (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Elizabeth A. Jolley (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802)
Abstract:
Many heat shock genes in bacteria are regulated through a class of
noncoding temperature sensitive RNAs, called RNA thermometers (RNATs).
The most widely studied RNAT is the ROSE (Repression Of heat Shock
Expression) element. ROSE elements are short RNA sequences that
contain two to four hairpin loops and are associated with expression of
small heat shock proteins. This project is focused on determining the effects
of the additional hairpins within ROSE elements on the functionality of the
RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that is well
known for containing multiple different ROSE elements. The 5’ UTR of the
ROSE element that encodes for heat shock protein A (hspA) in B.
japonicum has an intricate secondary structure containing three hairpins
upstream of the RNAT. While the RNAT within the 5’UTR of B. japonicum
has been previously studied, it is not clear how the upstream stem-loops
contribute to the temperature-sensing function of the ROSE elements. This
relationship was studied using UV thermal denaturation in various buffer
conditions. The individual hairpins and the full-length sequence were
studied and compared. A mutant RNA sequence was also created that
disrupted the stability of the stem-loop immediately upstream of the RNAT.
To determine if the role of the upstream hairpins and RNAT in B. japonicum
is evolutionarily important, other ROSE elements sequences were studied
computationally by bioinformatics. In each Mg2+ concentration, the WT
sequence showed several transitions within the melting curve indicating that
the RNA sequence is not folding cooperatively. When studied in the mutant
sequence, increasing Mg2+ concentration caused some transitions to
collapse to the same temperature range. The melt curve experiments
suggested that the upstream hairpins are important for stabilizing the RNAT,
and disruption of the immediately upstream stem-loop leads to decrease
thermal stability. Based upon the initial results of the bioinformatics study, it
appears as if there is increased thermal stability of the upstream hairpins
compared to the RNAT as observed within B. japoncium’s ROSE element.
Keywords: ROSE Elements, Secondary Structure