Gene machines

Abstract:

In this work we present a mathematical approach to model the kinetic behavior of DNA enzymes (DNAzymes) in order to predict their activity, which will assist future sequence designs. The model has been designed based on multiple previous reports since no general reaction mechanism has been fully described for DNAzymes to date. To better understand this, we also present first of its kind study of the DNAzyme catalytic reaction at the single molecule (SM) level.

Abstract:

Cotranscriptional RNA folding plays important roles in gene regulation steps such as splicing, transcription termination, and translation initiation. Progression of our understanding of cotranscriptional RNA folding mechanisms is still retarded by the lacking of experimental tools to study the kinetics of cotranscriptional RNA folding properly. In this chapter, we describe fluorescence resonance energy transfer (FRET) assay that enables the study of RNA cotranscriptional folding at the single-molecule level.

Abstract:

ABSTRACT

Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli . The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.