Abstract

Within each cell, genetic material is stored in the form of DNA. This DNA is compacted around proteins to form higher order structures called chromatin. The basic unit of chromatin is a histone protein octamer bound with DNA called a nucleosome. A nucleosome consists of eight core proteins, four pairs of histone proteins H2A, H2B, H3, and H4, wrapped by DNA. The DNA wraps around the histone protein roughly 1.7 times, helping facilitate the compaction of DNA within the nucleus of the cell. The location of nucleosomes makes genetic material that encodes various elements, such as promoters or enhancers, accessible or inaccessible to transcription machinery such as RNA polymerase II and other transcription factors. Thus, where and when nucleosomes are located across the DNA strand plays a major role in the three main steps of transcription: initiation, elongation, and termination. These histone proteins are frequently post-translationally modified. These modifications further play a role in transcription machinery accessibility to the DNA and how transcription can occur through nucleosome occupied areas. Transcription is a topic of prolific study and the various steps within transcription are well known. However, how nucleosome occupancy and various histone post-translational modifications affects the rate of transcription is a topic vastly understudied. To understand the various effects that histone post-translational modifications have on gene expression, most literature and past techniques use the final step in the central dogma, translation, to measure changes in gene expression rates, overlooking the first step in the central dogma, the step of transcription. To understand how the rate of transcriptional elongation changes with various nucleosomal post-translational modifications, we have developed a framework for an in vitro transcription study using a bacterial T7 promoter sequence coupled with a fluorescent aptamer. This framework allows for future studies in understanding how in vivo transcription rate is affected by nucleosomes and their modifications. This will be used as a framework so that further research can be done with chromatinized templates and with ex vivo C. elegans transcription extracts. An in vivo transcription system can be highly dynamic, and the addition or removal of various post-translational modifications can affect this rate. We observed through our framework that there are specific structures and various components needed to create an ideal reaction protocol for further application. We hypothesize that when our developed framework is applied to an in vivo system, histone post-translational modifications of methylation and acetylation will affect the rate, decreasing or increasing the rate of transcription respectively, as shown by a fluorescent aptamer. Thus, use of this fluorescent aptamer will allow better understanding of how histone modifications affect transcription.

Degree

College and Department

Life Sciences; Microbiology and Molecular Biology

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