Posts Tagged ‘histones’

Real-Time Visualization of DNA Transcription

 :: Posted by American Biotechnologist on 01-20-2011

Most of my graduate work focused on transcriptional regulation of a vasoregulatory gene and all the nitty-gritty work that goes along with these types of molecular protocols. As such, I am always on the lookout for techniques that improve upon current transcriptional regulation protocols especially if they show a propensity for doing the job either faster or better than is currently done.

In a study published today in Nature, L. Stirling Churchman et al from the University of California, San Fransisco reveal a technique that utilizes fast DNA-sequencing technology and advanced computer technology to examine transcriptional regulation with unprecedented resolution in real-time and in-vivo.

The native elongating transcript sequencing protocol, (NET-seq), is based on deep sequencing of 3′ ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution.

Until quite recently, many scientists thought that less than 5 percent of the human genome was actually transcribed into RNA and therefore used in the cell’s function, Churchman said. Recent advances in the field have revealed a tremendous complexity in that process, with new understanding that the majority of DNA is transcribed. Much of the product is still considered “junk RNA” – simply a byproduct of the process.

“Now, the question is not, ‘Why is that DNA there?’ but, ‘Why is that RNA there?’” said Churchman, a physicist and post-doctoral scholar at UCSF. “It could be junk RNA, but we don’t know.”

In the video below you can see how DNA is tightly wound around histones forming a nucleosome complex that is then packaged into the cell. Binding of transcriptional proteins to target DNA is not only contingent upon the DNA sequence but also depends upon the accessibility of the DNA template which depends upon how it is wrapped around the histone. As you can see in the video, some DNA remains accessible by virtue of the fact that it faces “outwards” while other portions of the DNA are harder for the transcriptional proteins to access.

In the present study, scientists were able to observe for the first time that polymerase comes in direct contact with the histone proteins during the transcription process, while also seeing how the nucleosomes acted as a speed bump for the polymerase enzyme as it moved along the genome transcribing DNA into RNA. In addition, the research showed that the organization of histone marks controlled whether “junk RNA” was produced from a given region of DNA.

Visit the UCSF site to read more.

Reference:
L. Stirling Churchman, & Jonathan S. Weissman (2011). Nascent transcript sequencing visualizes transcription at nucleotide resolution Nature, 469, 368-373 : doi:10.1038/nature09652