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ID 16471

Animation 21: RNA is an intermediary between DNA and protein.

Description:
Francis Crick describes RNA and its role and Paul Zamecnick explains protein synthesis.
Transcript:
Hello, I'm Francis Crick. The story of DNA does not end with Watson and me solving its three-dimensional structure. Because DNA is the hereditary molecule of the cell, we reasoned that the sequence of nucleotides in the molecule must function as a code, able to direct the synthesis of proteins. One puzzle we had to figure out was how DNA, which is found mainly in the cell's nucleus, can direct the synthesis of proteins that are made exclusively in the cell's cytoplasm. I proposed the "Central Dogma" where information flows from DNA to protein via a carrier molecule. A candidate for this information carrier was ribonucleic acid — RNA. RNA is a nucleic acid found mostly in the cell's cytoplasm. Like DNA, RNA has a sugar-phosphate backbone. However, RNA uses the sugar ribose instead of deoxyribose. DNA and RNA use the same nitrogenous bases except that DNA uses the nucleotide base thymine, whereas RNA uses uracil. Uracil can hydrogen bond with adenine just like thymine. DNA, as you know, is usually a double-stranded molecule. RNA, however, is usually single-stranded. After proposing the Central Dogma — where information flows from DNA to RNA to protein — I realized there was another problem. How did the amino acids interact with the carrier RNA? There must be adaptor molecules; in fact there must be 20 different adaptors, one for each amino acid. Hi, I'm Paul Zamecnik and I was interested in protein synthesis. In the 50's, I didn't know about Crick's Central Dogma or his adaptor hypothesis, and I approached the problem from a biochemical point of view. I made an extract using rat liver cells; it was basically a water-based solution that contained all the parts from a culture of cells. In 1953, I showed that this extract had everything needed to make proteins in a test tube. Remember that a protein is a polypeptide chain of amino acids linked to one another by peptide bonds. To follow protein construction, I added radiolabeled amino acids to the cell-free extract. After incubating the reaction at body temperature, I then centrifuged the tube. I expected that the newly-made polypeptides, being heavier, would pellet at the bottom of the tube. Unincorporated amino acids, being lighter, would remain in the supernatant. Sure enough, the radiolabel was present in the pellet, showing that new polypeptides had been synthesized. Mingled with the labeled polypeptides in the pellet, I identified a large cellular structure, later named the ribosome. It seemed clear the ribosome is the cytoplasmic organelle where protein assembly occurs. Ribosomes are constructed of both RNA and proteins. While the RNA part of the ribosome (rRNA) is involved in protein synthesis, its role was unclear. However, Mahlon Hoagland and I identified another RNA molecule associated with the unincorporated amino acids in the supernatant. Hi, I'm Mahlon Hoagland. I started working in Paul's lab in 1953, focusing on how amino acids were activated, or energized, prior to their incorporation into proteins. This is done by enzymes that I found, later known as aminoacylt RNA synthetases, in the soluble fraction of the cell. In 1955, Paul, Mary Stephensen and I found, to our surprise, that amino acids were first attached to a low molecular weight ("soluble") RNA in this same soluble fraction, and those amino acids were subsequently transferred to proteins in ribosomes. We named these intermediary carrier molecules "soluble" RNA. On a visit to our lab, Jim Watson recognized that soluble RNA met the requirements of Crick's adaptor hypothesis. Each of the soluble RNAs could pair to its partner amino acid and ferry the amino acid to the ribosome for protein synthesis. Soluble RNA was later renamed transfer RNA (tRNA) to better reflect its role. But, still, there was no answer to the problem of how the genetic code instructed cytoplasmic tRNAs and amino acids to make proteins. While some thought rRNA was the "template" on which proteins were built, it was becoming clear that rRNA did not have the right properties. So, now the hunt was on for the "information" molecule. But, still, there was no answer to the problem of how the genetic code instructed cytoplasmic tRNAs and amino acids to make proteins. While some thought rRNA was the "template" on which proteins were built, it was becoming clear that rRNA did not have the right properties. So, now the hunt was on for the "information" molecule. But, still, there was no answer to the problem of how the genetic code instructed cytoplasmic tRNAs and amino acids to make proteins. While some thought rRNA was the "template" on which proteins were built, it was becoming clear that rRNA did not have the right properties. So, now the hunt was on for the "information" molecule. Hello, I'm Sydney Brenner. With my colleagues, François Jacob and Matthew Meselson, I showed that rRNA was not the template for building proteins. There was a third type of RNA — an unstable intermediate — that carries the DNA message to the ribosome. We did this by using phage-infected bacteria. We started by growing bacteria in "heavy" isotopes of carbon and nitrogen to radiolabel all of the bacterial RNA and proteins. We then infected this bacterial culture with phage... …and immediately transferred the infected bacteria to media that lacked the heavy isotopes but contained radioactive 32P. We stopped phage growth before the bacteria were lysed and extracted RNA and ribosomes from the bacteria. We spun the bacterial extract in a density gradient in a centrifuge. This separated the various components and I was able to analyze the distribution of heavy and light isotopes in the bacterial ribosomes, and the incorporation of 32P in newly-made phage RNA. First, let's look at the ribosomes. One ribosome is made up of two subunits. In a density gradient, ribosomes can separate into two bands. The heavier band consists of whole ribosomes; the lighter band consists of dissociated subunits. I reasoned that if the rRNA in the ribosomes were the template for building new phage proteins, then new ribosomes with phage rRNA would have to be made after phage infection. As I suspected, this was not the case. All the ribosomes were made with heavy isotopes. Then I looked at where 32P had been incorporated in the production of new phage RNA. I found that 32P associated with the whole ribosome band and there was also 32P at the bottom of the tube. This turned out to be a new type of RNA. It was associated with the ribosomes so it must have a role in protein synthesis. However, it must be a large molecule since some of it was found in the sediment at the bottom of the tube. This was the information carrier envisioned by Crick in his Central Dogma, and I named it messenger RNA (mRNA).
Keywords:
sugar phosphate backbone, carrier rna, central dogma, francis crick, protein synthesis, hydrogen bond, amino acids, ribonucleic acid
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