Trends in Biotechnology Molecules of Genetics. Concept 15 - DNA and proteins are key molecules of the cell nucleus. DNA was discovered as a major chemical

Trends in Biotechnology

Molecules of Genetics

Concept 15 - DNA and proteins are key molecules of the cell nucleus.

DNA was discovered as a major chemical of the nucleus at about the same time Mendel and Darwin published their work. However, during the early 1900s, more people thought that proteins were the molecules able to carry large amounts of hereditary information from generation to generation.

Although DNA was known to be a very large molecule, it seemed likely that its four chemical components were assembled in a monotonous pattern — like a synthetic polymer. Also, no specific cellular function had yet been found for DNA.

Proteins, on the other hand, were important as enzymes and structural components of living cells. Proteins were also known to be polymers of numerous amino acids. These polymers are called polypeptides. Most importantly, the 20 amino acid "alphabet" of proteins potentially could be configured into more unique information-carrying structures than the four-letter alphabet of DNA.

A nucleotide is made of three elements: phosphate, deoxyribose sugar, and a nitrogenous base.

The carbons of the deoxyribose sugar are numbered 1-5. In a nucleotide, the noitrogenous base is always bound to carbon#1, a hydroxyl group (OH) is bound to carbon#3 and the phosphate group is bound to carbon#5.

Each of the four nucleotides has a distinct nitrogenous base.

The sugars are connected to the phosphate group through a phosphodiester bond.

The phosphodiester bonds give the molecule a direction; from carbon#5 to carbon#3. [ 5 prime (5’) to 3 prime (3’) ]

• Animation at http://www.dnaftb.org/15/animation.html

• The review problem is at http://www.dnaftb.org/15/problem.html

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http://www.dnaftb.org/15/problem.html

Concept 16 - One gene makes one protein.

In 1902, Archibald Garrod described the inherited disorder alkaptonuria as an "inborn error of metabolism." He proposed that a gene mutation causes a specific defect in the biochemical pathway for eliminating liquid wastes. The phenotype of the disease — dark urine — is a reflection of this error.

Strong evidence for this hypothesis was found in 1941 by George Beadle and Edward Tatum, using the simple bread mold Neurospora. First, they found that molds exposed to radiation lose the ability to produce essential nutrients, and this slowed, even stopped the growth of the mold. Then, they found that growth can be restored by providing the mutated mold with a specific supplement. They reasoned that each mutation must inactivate the enzyme (protein) needed to synthesize the nutrient. Thus, one gene carries the directions for making one protein.





Concept 17 - A gene is made of DNA.

In the 1920s, experiments showed that a harmless strain of bacteria can become infectious when mixed with a virulent strain of bacteria that had been killed. The dead bacteria apparently provide some chemical that "transforms" the harmless bacteria to infectious ones. This so-called "transforming principle" appeared to be a gene.

A team of scientists led by Oswald Avery, followed up on these experiments in the 1940's. They found that a pure extract of the "transforming principle" was unaffected by treatment with protein-digesting enzymes but was destroyed by a DNA-digesting enzyme. This showed that the transforming principle is DNA — and, by extension, a gene is made of DNA. Still, many scientists were slow to accept this clear proof that DNA, not protein, is the genetic molecule.





Concept 18 - Bacteria and viruses have DNA too.

Microscopes proved the existence of single-celled bacteria. However, there was debate about whether bacteria had genes and what attributes they may have in common with higher life forms. This debate was settled in the 1940's, when it was discovered that bacteria have sex. During the process of conjugation, genes are exchanged through a mating channel that links two bacteria.

Electron microscopy suggested that bacterial viruses carry on a similar process. A virus attaches to a host bacterium and injects its genes through its channel-like tail. In 1952, Alfred Hershey showed that DNA, alone, is responsible for the reproduction of new viruses within an infected cell. This provided undeniable support for Avery's earlier experiments that a gene is made of DNA. It also showed that viruses, as well as bacteria, can be used as models for studying universal principles of genetics.





Concept 19 - The DNA molecule is shaped like a twisted ladder.Earlier work had shown that DNA is composed of building blocks called nucleotides consisting of a deoxyribose sugar, a phosphate group, and one of four nitrogen bases — adenine (A), thymine (T), guanine (G), and cytosine (C). Phosphates and sugars of adjacent nucleotides link to form a long polymer. Other key experiments showed that the ratios of A-to-T and G-to-C are constant in all living things. X-ray crystallography provided the final clue that the DNA molecule is a double helix, shaped like a twisted ladder.

In 1953, the race to determine how these pieces fit together in a three-dimensional structure was won by James Watson and Francis Crick at the Cavendish Laboratory in Cambridge, England. They showed that alternating deoxyribose and phosphate molecules form the twisted uprights of the DNA ladder. The rungs of the ladder are formed by complementary pairs of nitrogen bases — A always paired with T and G always paired with C.





Concept 20 - A half DNA ladder is a template for copying the whole.Because of the pairing of adenine-to-thymine and guanine-to-cytosine, Watson and Crick proposed that one half of the DNA ladder can be a template for recreating the other half during DNA replication. By 1958, two lines of evidence came together to provide proof of this hypothesis. First, an enzyme was discovered — DNA polymerase — that adds complementary nucleotides to the template provided by a half DNA molecule.

Second, an ingenious experiment used nitrogen isotopes to follow the construction of new DNA molecules during successive generations of bacteria. This showed that one strand of each DNA molecule is passed along unchanged to each of two daughter cells. This "conserved" strand acts as the template for DNA polymerase to synthesize a second complementary strand, which completes each new DNA molecule.





Concept 21- RNA is an intermediary between DNA and protein.DNA is found mostly in the cell nucleus, but another type of nucleic acid, RNA, is common in the cytoplasm. Watson and Crick proposed that RNA must copy the DNA message in the nucleus and carry it out to the cytoplasm, where proteins are synthesized. Crick also predicted the existence of an "adaptor" molecule that reads the genetic code and selects the appropriate amino acids to add to a growing polypeptide chain. This proposed flow of genetic information from DNA to RNA to protein became known as the "Central Dogma."

As it turned out, several types of RNA are involved in the utilization of genetic information. In the nucleus, the DNA code is "transcribed," or copied, into a messenger RNA (mRNA) molecule. In the cytoplasm, the mRNA code is "translated" into amino acids. Translation is orchestrated at the ribosome — itself partly composed of RNA — with transfer RNA playing the role of adaptor.





Information flows between DNA, RNA and protein. DNA -> protein is another special transfer, but it is not found in nature.

Concept 22 - DNA words are three letters long.

The genetic code had to be a "language" — using the DNA alphabet of A, T, C, and G — that produced enough DNA "words" for each of the 20 known amino acids. Only 16 words are possible from a two-letter combination, but a three-letter code produces 64 words. Researchers assumed a three-letter code called a codon.

Research teams carefully synthesized different RNA molecules, each a long strand of a single repeated codon. Then, each type of synthetic RNA was added to a cell-free translation system containing ribosomes, transfer RNAs, and amino acids. Each type of synthetic RNA produced a polypeptide chain of repeated units of a single amino acid. Several codons are "stop" signals and many amino acids are specified by several different codons. All 64 three-letter combinations do something.

In 1961, Marshall Nirenberg and J. H. Matthaei published their paper. They showed that a synthetic messenger RNA made of only uracils can direct protein synthesis. The polyU mRNA resulted in a poly-phenylalanine protein They had the first piece of the genetic code.

Later, Nirenberg and his group found the entire genetic code by matching amino acids to synthetic triplet nucleotides. There is redundancy (some amino acids are encoded by more than one codon) and some codons are "punctuation marks" in the mRNA message. They also showed that with few exceptions, the genetic code was the same for all organisms.





Concept 23 - A gene is a discrete sequence of DNA nucleotides.Mendel described a gene as a discrete unit of heredity that influences a visible trait. Beadle and Tatum defined a gene as the discrete directions for making a single protein, which influences a metabolic trait. Early sequencing efforts showed that proteins are, in turn, long chains of amino acids arranged in a specific order. The triplet genetic code further refined the definition of a gene as a discrete sequence of DNA encoding a protein — beginning with a "start" codon and ending with a "stop" codon.

Gene analysis took a giant step forward with the discovery of methods to determine the exact sequence of nucleotides that compose a specific gene. DNA sequencing was built upon earlier knowledge of DNA polymerases and cell-free systems for replicating DNA. The chain-termination method, which makes clever use of a "defective" DNA nucleotide, now dominates DNA sequencing technology.





Concept 24 - The RNA message is sometimes edited.Dogma and logic dictated that the mRNA code is a faithful representation of the DNA from which it is transcribed. This exact correspondence between mRNA sequence and DNA sequence was generally upheld in experiments with bacterial cells (prokaryotes).

However, inconsistencies surfaced as recombinant-DNA techniques allowed researchers to explore the genes of higher cells (eukaryotes). Then, it was found that mRNA transcripts appeared to be shorter than their corresponding genes. This difference became obvious in electron micrographs of mRNA bound to its complementary DNA template — where regions of DNA without corresponding mRNA form loops.

In fact, the protein coding information in genes is interrupted by non-coding sequences called introns, which results in "split genes." The entire DNA code is faithfully transcribed into a temporary form of RNA (pre-mRNA), but this is edited in the nucleus to yield a mature mRNA. The process of RNA splicing involves removing non-coding regions, introns, and splicing together adjacent coding regions, exons.





Concept 25 - Some viruses store genetic information in RNA.DNA was believed to be the sole medium for genetic information storage. Furthermore, Watson and Crick's central dogma assumed that information flowed "one-way" from DNA to RNA to protein. So it came as a surprise when in 1971, it was discovered that some viruses shift their genetic information from RNA to DNA.

Even so, these viruses ultimately make proteins in the same way as higher organisms. During infection, the RNA code is first transcribed "back" to DNA — then to RNA to protein, according to the accepted scheme. The initial conversion of RNA to DNA — going in reverse of the central dogma — is called reverse transcription, and viruses that use this mechanism are classified as retroviruses. A specialized polymerase, reverse transcriptase, uses the RNA as a template to synthesize complementary and double-stranded DNA molecule.





Concept 26 - RNA was the first genetic molecule.Experiments in the 1960s showed that messenger RNA has the ability to store genetic information, while transfer and ribosomal RNA have the ability to translate genetic information into proteins. Experiments performed two decades later showed that some RNAs can even act as an enzyme to self-edit their own genetic code!

These results raised two questions: 1) Why does RNA play so many roles in the flow of genetic information? 2) Why bother storing genetic information in DNA, if RNA alone could do the job?

RNA has great capability as a genetic molecule; it once had to carry on hereditary processes on its own. It now seems certain that RNA was the first molecule of heredity, so it evolved all the essential methods for storing and expressing genetic information before DNA came onto the scene. However, single-stranded RNA is rather unstable and is easily damaged by enzymes. By essentially doubling the existing RNA molecule, and using deoxyribose sugar instead of ribose, DNA evolved as a much more stable form to pass genetic information with accuracy.







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Trends in Biotechnology Molecules of Genetics. Concept 15 - DNA and proteins are key molecules of the cell nucleus. DNA was discovered as a major chemical