Undergraduate Honors Theses

Thesis Defended

Spring 2016

Document Type

Thesis

Type of Thesis

Departmental Honors

Department

Biochemistry

First Advisor

Robert Batey

Second Advisor

Joseph Falke

Third Advisor

Thomas Perkins

Abstract

Riboswitches are gene regulatory elements found in the 5’-untranslated region of many bacterial genes. The direct binding of small molecule ligands induces conformational changes in the RNA that influence downstream expression machinery and determine the transcription or translation of an attached gene. Cobalamin riboswitches, which bind coenzyme-B12 and other cobalamin variants, are divided into two distinct classes. Cbl-I riboswitches contain a large peripheral element that limits riboswitch binding to only coenzyme-B12 (adenosylcobalamin, or AdoCbl). Cbl-II riboswitches do not contain the same peripheral element, and were originally thought to only bind smaller species of cobalamin (including methylcobalamin, or MeCbl). However, a sample of Cbl-II variants reveals a range of riboswitch binding affinities to both AdoCbl and MeCbl, with some RNAs binding both species of cobalamin with equal affinity. In order to determine the structural components of the Cbl-II riboswitches that are important in establishing ligand selectivity, a series of RNA mutants were designed to convert an AdoCbl-selective riboswitch (WTenv50) to MeCbl-selectivity. Sequences from the J6/3 and J3/4 binding core, the J1/3 peripheral element, and the P4/P6 interaction were targeted, using a MeCbl-specific riboswitch (WTenv8) as a guide. Isothermal titration calorimetry (ITC) results indicate that the J1/3 interaction with J6/3 is critical in conferring ligand selectivity in Cbl-II riboswitches. Ongoing experimentation on WTenv8 scaffold mutants with WTenv50 directed mutations might yield further insight into Cbl-II riboswitch ligand selectivity and into broader RNA strategies for general ligand binding.