User:Anthony Salvagno/Notebook/Research/2009/10/01/GRD Proposal 2009

DNA is the genetic information contained inside in the cells of every living organism. This information is important because it is the code that makes each individual organism unique. It is the expression of DNA (the conversion of DNA into proteins) that determines what a person may look like, how much muscle a lion can have, have long a giraffe's neck can be, how lethal the next flu strain can be, etc.

DNA is a double stranded polymer. Each strand is made of a sugar backbone with four distinct nucleotides that allow two strands to join in a helix conformation. Those bases are cytosine, guanine, thymine, and adenine. Each base plays an important role in the structure of DNA because it is the sequence of these bases that determine one's genetic code. In humans there are 23 chromosomes, each full of tightly compacted DNA, and each chromosome has a number of genes. A gene is a section of DNA that provides information about the makeup of a specific cellular component. There are literally tens of thousands of genes in the human genome (the entirety of all the DNA of an organism).

The process by which DNA becomes a protein is a very complex process. It begins with transcription, where an RNA Polymerase (RNA Polymerase II in humans, or RNA Pol II) enzyme reads a single strand of DNA and creates a complementary strand of messenger RNA (mRNA) from it. This single stranded RNA polymer may then undergo a few changes so that it can be read by complementary tRNA during an event known as translation. Each tRNA molecule (consisting of 3 bases) comes attached with a specific amino acid and the amino acids form long chains known as polypeptides. The sequence of mRNA (which is determined by DNA) determines the sequence of tRNA which then determines the amino acid order. These polypeptides undergo a folding process and the result is a protein which the cell can use for a variety of functions, all of which are crucial to life.

I am interested in a specific part of this long, complicated process and that is the behavior of RNA Pol II during transcription. In order for transcription to begin, various proteins bind to the DNA in order to build the Pol II complex (the polymerase enzyme is itself a complex of proteins that work together). Once the Pol II enzyme is constructed, it will attempt to begin reading the DNA to create complementary mRNA. In order for this to occur, it must be constructed at the correct location on a DNA chain known as the promoter. The promoter marks the beginning of a gene. Once RNA Pol II reaches the end of a gene, transcription can end so that the newly formed transcript (the new mRNA molecule) can be sent off for translation.

This entire process is very complicated, and if at any point there is an error in the process there can be disastrous results. In order for proteins vital to cell life to function properly transcription must proceed effeciently and correctly. I would like to be able to study various properties of the RNA Polymerase II molecule with the use of Optical Tweezers (OT). With OT, I can study individual RNA Pol II molecules under certain conditions. I would be able to study properties during construction of the complex, during the transcription event, during termination (end of transcription), and provide never-before-seen glimpses into the behavior of this important enzyme.