Poster abstracts

Poster number 58 submitted by Angela Smith

Molecular basis for thiamin pyrophosphate recognition and gene regulation by the THI-box riboswitch

Angela M. Smith (Department of Microbiology, The Ohio State University), Nancy Ontiveros-Palacios, Mario Soberon (Instituto de Biotecnologίa, Universidad Nacional Autόnoma de Mexico), Frank J. Grundy, Tina M. Henkin (Department of Microbiology, The Ohio State University), Juan Miranda-Rios (Instituto de Biotecnologίa, Universidad Nacional Autόnoma de Mexico)

Abstract:
Riboswitches are RNA elements that modulate expression of genes upon direct sensing of specific regulatory signals. These elements respond to a wide range of environmental signals such as temperature change, small molecules, and non-coding RNAs. The THI-box riboswitch regulates expression of genes involved in thiamin biosynthesis in response to thiamin pyrophosphate (TPP). In E. coli the thiM gene is regulated at the level of translation initiation by a THI-box riboswitch located in the 5’ leader of the mRNA. In the TPP-bound form, the RNA folds into a structure in which the Shine-Dalgarno (SD) sequence is sequestered through base pairing with an anti-SD (ASD) sequence. When TPP levels are low, the ASD sequence forms an alternate pairing with an upstream sequence, the anti-anti-SD (AASD), leaving the SD region accessible for ribosome binding. An in vivo expression analysis using translational fusions to a lacZ reporter was used to investigate the effects of TPP binding on gene expression using the wild-type THI-box RNA and mutant constructs containing nucleotide substitutions in the TPP-binding domain. Mutation of residues predicted to be involved in TPP binding resulted in a loss of TPP-dependent repression with two distinct phenotypes observed. Class I mutations resulted in constitutive expression regardless of the presence of thiamin, while Class II mutations resulted in constitutive repression. In vitro methods were used to determine the effect of these nucleotide changes on TPP-dependent conformational changes at the SD region. Results from these experiments suggest that the SD region of Class I mutant constructs is accessible regardless of the presence of TPP. However, in Class II mutant constructs, the RNA favors a closed conformation in which accessibility of the SD region is limited, resulting in inhibition of translation regardless of the presence of TPP.

Keywords: THI-box, riboswitch, regulation