Bio 100 - Molecular Genetics 1 A. Bacterial Transformation Chapter 10 - Molecular Biology of the Gene Researchers found that they could transfer an inherited characteristic (e.g. the ability to cause pneumonia), from one strain of bacteria to another, by exposing a harmless bacteria strain to DNA extracted from a disease causing strain This process of transferring an inherited trait by an extract of DNA is called transformation B. Bacterial Invaders Definitive proof of the gene-dna connection came from work with bacteriophages ("bacteria eaters") which are viruses consisting of DNA surrounded by a protein shell They infect bacteria cells causing them to make more phages: Q. What commandeered this? Protein of DNA Alfred Hershey and Martha Chase Experiment Exposed E. coli bacteria with phages whose DNA and protein were labeled by radioactive compounds that could be distinguished from one another They found that the phage DNA entered the bacteria but the protein did not The Structure of DNA In 1951, James Watson and Francis Crick attempted to build a model that would show the molecular structure of DNA that until this time was largely unknown What was known about DNA at that time: 1. DNA is a polymer of nucleotides, each one having a phosphate group, the sugar deoxyribose, and a nitrogenous base 2. Erwin Chargaff, formulated Chargaff s Rules: The number of purines in DNA always equals the number of pyrimidines The amount of adenine equals the amount of thyamine the amount of cytosine equals the amount of guanine
Bio 100 - Molecular Genetics 2 3. Rosalind Franklin and Maurice Wilkins had prepared an X-ray defraction photograph of DNA indicating that DNA was long and skinny, helical and consisted of 2 parallel strands The model proposed by Watson and Crick suggested the following features of DNA: 1. The DNA molecule is composed of 2 nucleotide chains oriented in opposite directions In each strand each P group is attached to one sugar at the 5 position (the 5th C) and to the other at the 3 position 2. The DNA molecule is analogous to the structure of a ladder The bases on the 2 strands are directed inward and form the rung of the ladder, while the sugar-phosphate face outside and act as the sides of the ladder 3. Complimentary base pairing - bases A and T always paired and G and C always paired 4. They suggested that the two DNA strands are twisted together to form intertwinned helices = double helix The Structure of RNA The pentose sugar in RNA is ribose The pyrimidine thyamine is replaced with uracil Thus, the 4 bases are: A, U, C, and G RNA is single stranded, and does not form the double helix like DNA There are 3 different classes of RNA: mrna, rrna, and trna DNA has 2 primary functions: 1. To replicate the stored information it contains 2. Use the stored information in the synthesis of proteins Central Dogma of molecular genetics: DNA ---------------------------> RNA --------------------> protein transcription translation
Bio 100 - Molecular Genetics 3 DNA Replication: A Closer Look The double stranded DNA molecule lends itself to replication because each strand can serve as a template for the formation of a complimentary strand DNA replication requires the following steps: 1. The double helix unwinds and unzips The unwinding is largely due to the activity of helicase Unzipping occurs at a distinct site along the chromosome and creates a replication bubble The y-shaped junction at the spot where the 2 strands are unwinding is called the replication fork 2. Complimentary Base Pairing The exposed sequence of bases are paired with free nucleotides that drift into the area 3. Joining Base pairs are joined together in a process called polymerization It is catalyzed by DNA polymerase Note: DNA polymerase moves in one direction on the DNA (e.g. toward the replication fork) Thus nucleotides are added to the newly formed DNA chain in the 5-3 direction DNA replication is continuous on this side of the template DNA replication is discontinuous on the other template An enzyme, ligase, will later link these stretches In the end you get 2 double stranded DNA molecules, identical to one another; replication is semiconservative Accuracy of the Replication In some cases, bases pairs do not pair correctly with one another during replication Nonetheless, the error rate is minimal because DNA polymerase has a "proofreading" function On average there is one mistake per billion nucleotides, or 3 mistakes per mitosis
Bio 100 - Molecular Genetics 4 Transcription (DNA ----> RNA) During transcription, the DNA code is passed to mrna 1. A segment of the DNA helix unwinds and unzips, and complimentary RNA nucleotides pair with the DNA nucleotides of one strand 2. Initiation - RNA nucleotides are joined by the enzyme RNA polymerase, and the mrna molecule is formed RNA polymerase must be must be instructed where to start and where to stop the transcribing process The start transcribing signal is a nucleotide sequence called a promoter located in the DNA next to the beginning of the gene 3. After initiation, the RNA (mrna) strand is elongates Finally, there is termination where the RNA polymerase reaches a special sequence of bases in the DNA template called the terminator RNA Processing In eukaryotes, after the mrna strand is formed, it passes from the nucleus to the cytoplasm where it becomes associated with ribosomes But, before it leaves the nucleus, the mrna is modified or processed One kind of processing is the addition of extra nucleotides (The cap and tail ) to the ends of the RNA transcript Another type of processing results from the fact that DNA is comprised of long stretches of noncoding regions (=introns) that interrupt nucleotide sequences that code for amino acids The coding regions - the parts of the gene that are expressed - are called exons Both exons and introns are transcribed from DNA into RNA RNA splicing - before the RNA leaves the nucleus, the introns are removed and the exons are joined to produce the mrna with a continuous coding sequence
Bio 100 - Molecular Genetics 5 Translation (mrna -----> protein) During translation the sequence of bases of the mrna is translated into a sequence of amino acids 3 nucleotide units of the mrna that codes for a amino acid is called a codon e.g., CGU codes for the aa Arginine Also, there are 3 codons called stop codons, that serve as a signal polypeptide chain termination e.g., UAA, UAG, UGA One codon is a start codon that signals to begin polypeptide e.g., AUG Translation: A Closer Look During translation, the sequence of codons in mrna dictates the order of the amino acids in the polypeptide Translation requires the efforts of several enzymes, and 2 other types of RNA: rrna and trna rrna It makes up the ribosomes; they consist of 2 subunits, each of which is comprised of rrna and proteins rrna is transcribed from DNA in the area of the nucleolus Ribosomes function in coordinating protein synthesis Each ribosome has a binding site for mrna on its small subunit Its large subunit serves as a binding sites for trna: the P site holds the trna carrying the growing polypepetide chain; the A site holds a trna carrying the next amino acid to be added to the chain trna trna is also transcribed in the nucleus and later moves to the cytoplasm trna brings amino acids from the cytoplasm to the ribosomes located in the cytoplasm Amino acids attach to one end of the trna molecule (CCA sequence) The other end of the molecule has a codon complimentary to the mrna codon and is called the anticodon
Bio 100 - Molecular Genetics 6 There are 3 important steps in translation: initiation, elongation, and termination 1. Initiation The mrna molecule binds (5 end) to a small ribosomal subunit Initiation begins at the area of the start codon (AUG) of m RNA The first trna pairs with the start codon of mrna A large ribosomal subunit then attaches to the smaller unit This completes the ribosomal structure It possess 2 active sites of attachment for trna 2. Elongation The ribosome moves down the length of the mrna (5' ---> 3') It stops at the second and third mrna codons Two trnas are then received by these codons, each carrying a specific amino acid The ribosome moves again, occupying the third and fourth codon, etc. As the ribosome moves past an mrna codon with a trna, the trna are released and re-enter the cytoplasms trna pool When each of the trnas with an amino acid are attached to the ribosome, the enzyme peptidyl transferase forges a peptide bond between 2 amino acids 3. Termination The process continues until the ribosome encounters one of three possible stop codons e.g. UAG, UAA, UGA The is no trna to recognize these codons; instead a release factor will bind to the stop codon The last trna is released and the polypetide chain is freed The polypeptides may then go to the ER where further synthesis occurs (e.g. secondary, tertiary structure) Some goes to the cellular organelles e.g. mitochondria and chloroplasts