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Posts Tagged ‘Transcription (genetics)’


RNA polymerase – molecular basis for DNA transcription

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E: 2; 3.1

Roger Kornberg, MD
Nobel Prize in Chemistry
Stanford University

Son of Arthur Kornberg, who received the Nobel Prize for DNA polymerase, Roger Kornberg spent decades on the problem of transcription of the genetic code in eukaryotic cells. Roger Kornberg made several contributions to the understanding of the transcription model including – recognition of the nucleosomal structure of DNA, characterization of the chromatin modifying factors, and discovering the bridging factor that mediates transcriptional activation (called Mediator). The three types of RNA are termed mRNA, tRNA, and rRNA. Kornberg recognized that chromatin consists of nucleosomes arranged along DNA in the form of beads on a string. He used electron crystallography to determine that lateral diffusion in molecules tethered to the bilayer to pack into two-dimensional crystals suitable for crystallography.   Using yeast, Kornberg identified the role of RNA polymerase II and other proteins in transcribing DNA, and he created three-dimensional images of the protein cluster using X-ray crystallography. Polymerase II is used by all organisms with nuclei, including humans, to transcribe DNA.

While a graduate student working with Harden McConnell at Stanford in the late 1960s, he discovered the “flip-flop” and lateral diffusion of phospholipids in bilayer membranes. While a postdoctoral fellow working with Aaron Klug and Francis Crick at the MRC in the 1970s, Kornberg discovered the nucleosome as the basic protein complex packaging chromosomal DNA in the nucleus of eukaryotic cells (chromosomal DNA is often termed “Chromatin” when it is bound to proteins in this manner, reflecting Walther Flemming‘s discovery that certain structures within the cell nucleus would absorb dyes and become visible under a microscope).[10] Within the nucleosome, Kornberg found that roughly 200 bp of DNA are wrapped around an octamer of histone proteins.

Kornberg’s research group at Stanford later succeeded in the development of a faithful transcription system from baker’s yeast, a simple unicellular eukaryote, which they then used to isolate in a purified form all of the several dozen proteins required for the transcription process. Through the work of Kornberg and others, it has become clear that these protein components are remarkably conserved across the full spectrum of eukaryotes, from yeast to human cells.

Using this system, Kornberg made the major discovery that transmission of gene regulatory signals to the RNA polymerase machinery is accomplished by an additional protein complex that they dubbed Mediator.[11] As noted by the Nobel Prize committee, “the great complexity of eukaryotic organisms is actually enabled by the fine interplay between tissue-specific substances, enhancers in the DNA and Mediator. The discovery of Mediator is therefore a true milestone in the understanding of the transcription process.”[12]

Kornberg took advantage of expertise with lipid membranes gained from his graduate studies to devise a technique for the formation of two-dimensional protein crystals on lipid bilayers. These 2D crystals could then be analyzed using electron microscopy to derive low-resolution images of the protein’s structure. Eventually, Kornberg was able to use X-ray crystallography to solve the 3-dimensional structure of RNA polymerase at atomic resolution.[13][14] He extended these studies to obtain structural images of RNA polymerase associated with accessory proteins.[15] Through these studies, Kornberg created an actual picture of how transcription works at a molecular level.

“I measured the molecular weight of the purified H3/H4 preparation by equilibrium ultracentrifugation, while Jean Thomas offered to analyze the material by chemical cross-linking. Both methods showed unequivocally that H3 and H4 were in the form of a double dimer, an (H3)2(H4)2 tetramer (Kornberg and Thomas, 1974). I pondered this result for days, and came to the following conclusions (Kornberg, 1974). First, the exact equivalence of H3 and H4 in the tetramer implied that the differences in relative amounts of the histones from various sources measured in the past must be due to experimental error. This and the stoichiometry of the tetramer implied a unit of structure in chromatin based on two each of the four histones, or an (H2A)2(H2B)2(H3)2(H4)2 octamer. Second, since chromatin from all sources contains roughly one of each histone for every 100 bp of DNA, a histone octamer would be associated with 200 bp of DNA. Finally, the (H3)2(H4)2 tetramer was reminiscent of hemoglobin, an a2b2 tetramer. The X-ray structures of hemoglobin and other oligomeric proteins available at the time were compact, with no holes through which a molecule the size of DNA might pass. Rather, the DNA in chromatin must be wrapped on the outside of the histone octamer.

As I turned these ideas over in mind, it struck me how I might explain the results of Hewish and Burgoyne. What if their sedimentation coefficient of unit length DNA fragments was measured under neutral rather than alkaline conditions? Then the DNA would have been double stranded and about 250 bp in length. Allowing for the approximate nature of the result, the correspondence with my prediction of 200 bp was electrifying. Then I recalled a reference near the end of the Hewish and Burgoyne paper to a report of a similar pattern of DNA fragments by Williamson. I rushed to the library and found that Williamson had obtained a ladder of DNA fragments from the cytoplasm of necrotic cells and measured the unit size by sedimentation under neutral conditions: the result was 205 bp! … with colleagues in Cambridge, I proved the existence of the histone octamer and the equivalence of the 200 bp unit with the particle seen in the electron microscope (Kornberg, 1977). This chapter of the chromatin story concluded with the X-ray crystal structure determination of the particle, now known as the nucleosome, showing a histone octamer surrounded by DNA, in near atomic detail (Luger et al., 1997).

I had decided to pursue the function rather than the structure of the nucleosome, and was joined in this by Yahli Lorch, who became my lifelong partner in chromatin research, and also my partner in life. We investigated the consequences of the nucleosome for transcription. It was believed that histones are generally inhibitory to transcription. We found, to the contrary, that RNA polymerases are capable of reading right through a nucleosome. Coiling of promoter DNA in a nucleosome, however, abolished initiation by RNA polymerase II (pol II) (Lorch et al., 1987). This finding, together with genetic studies of Michael Grunstein and colleagues, identified a regulatory role of the nucleosome in transcription. It has since emerged that nucleosomes play regulatory roles in a wide range of chromosomal transactions. A whole new field has emerged, one of the most active in bioscience today. It involves a bewildering variety of posttranslational modifications of the histones, and a protein machinery of great complexity for applying, recognizing, and removing these modifications.”

 

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Larry H Bernstein, MD, FCAP, Reporter

Long noncoding RNA (lncRNA) lightens up the dark secrets

CASE WESTERN RESERVE INVESTIGATORS DISCOVER NOVEL CELLULAR GENES BY UNCOVERING UNCHARACTERIZED RNAS THAT ENCODE PROTEINS

News Release: June 23, 2014

Jeannette Spalding
216-368-3004
jeannette.spalding@case.edu 

Case Western Reserve School of Medicine scientists have made an extraordinary double discovery. First, they have identified thousands of novel long non-coding ribonucleic acid (lncRNA) transcripts. Second, they have learned that some of them defy conventional wisdom regarding lncRNA transcripts, because they actually do direct the synthesis of proteins in cells.

Both of the breakthroughs are detailed in the June 12 issue of Cell Reports.

Kristian E. Baker, PhD, assistant professor in the Center for RNA Molecular Biology, led the team that applied high throughput gene expression analysis to yield these impressive findings, which ultimately could lead to treatments for cancer and some genetic disorders.

“Our work establishes that lncRNAs in yeast can encode proteins, and we provide evidence that this is probably true also in mammals, including humans,” Baker said. “Our investigation has expanded our knowledge of the genetic coding potential of already well-characterized genomes.”

Collaborating with researchers including Case Western Reserve University graduate and undergraduate students, Baker analyzed yeast and mouse cells, which serve as model organisms because of their functional resemblance to human cells.

Previously, lncRNAs were thought to lack the information and capacity to encode for proteins, distinguishing them from the messenger RNAs that are expressed from known genes and act primarily as templates for the synthesis of proteins. Yet this team demonstrated that a subset of these lncRNAs is engaged by the translation machinery and can function to produce protein products.

In the future, Baker and fellow investigators will continue to look for novel RNA transcripts and also search for a function for these lncRNAs and their protein products in cells.

“Discovery of more transcripts equates to the discovery of new and novel genes,” Baker said. “The significance of this work is that we have discovered evidence for the expression of previously undiscovered genes. Knowing that genes are expressed is the very first step in figuring out what they do in normal cellular function or in dysfunction and disease.”

This investigation was funded by the National Institutes of Health’s National Institute of General Medical Sciences (GM080465 and GM095621) and the National Science Foundation (NSF1253788).

 

Reference:

Lecture Contents delivered at Koch Institute for Integrative Cancer Research, Summer Symposium 2014: RNA Biology, Cancer and Therapeutic Implications, June 13, 2014 @MIT

Curator of Lecture Contents: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.wordpress.com/wp-admin/post.php?post=23174&action=edit
3:15 – 3:45, 6/13/2014, Laurie Boyer “Long non-coding RNAs: molecular regulators of cell fate”    

 https://pharmaceuticalintelligence.com/2014/06/13/315-345-2014-laurie-boyer-long-non-coding-rnas-molecular-regulators-of-cell-fate/

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Cardiac Ca2+ Signaling: Transcriptional Control

Reporter: Larry H Bernstein, MD, FCAP

The other side of cardiac Ca2+ signaling: transcriptional control

A Domínguez-Rodríguez, G Ruiz-Hurtado, Jean-Pierre Benitah and AM Gómez

  • Ca2+ is not only a key element in excitation-contraction coupling (EC coupling), but
  • it is also a pivotal second messenger in cardiac signal transduction,
  • being able to control processes such as
    • excitability,
    • metabolism, and
    • transcriptional regulation.

Front. Physio. 2012; 3:452.                 http://dx.doi.org/fphys.2012.00452/
http://www.fphys.com/The other side of cardiac Ca2+ signaling: transcriptional control

calcium release calmodulin

calcium release calmodulin

English: A rendition of the CaMKII holoenzyme ...

English: A rendition of the CaMKII holoenzyme in the (A) Closed and the (B) Open conformation (Photo credit: Wikipedia)

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