Chapter 10 - Molecular Biology of the Gene

Bio 100 - Molecular Genetics 1A. Bacterial TransformationChapter 10 - Molecular Biology of the GeneResearchers found that they could transfer an inherited characteristic (e.g. the ability tocause pneumonia), from one strain of bacteria to another, by exposing a harmless bacteriastrain to DNA extracted from a disease causing strainThis process of transferring an inherited trait by an extract of DNA is calledtransformationB. Bacterial InvadersDefinitive proof of the gene-DNA connection came from work with bacteriophages("bacteria eaters") which are viruses consisting of DNA surrounded by a protein shellThey infect bacteria cells causing them to make more phages:Q. What commandeered this? Protein of DNAAlfred Hershey and Martha Chase ExperimentExposed E. coli bacteria with phages whose DNA and protein were labeled byradioactive compounds that could be distinguished from one anotherThey found that the phage DNA entered the bacteria but the protein did notThe Structure of DNAIn 1951, James Watson and Francis Crick attempted to build a model that would show themolecular structure of DNA that until this time was largely unknownWhat was known about DNA at that time:1. DNA is a polymer of nucleotides, each one having a phosphate group, the sugardeoxyribose, and a nitrogenous base2. Erwin Chargaff, formulated Chargaff’s Rules:The number of purines in DNA always equals the number of pyrimidinesThe amount of adenine equals the amount of thyamine the amount of cytosine equals theamount of guanine

Bio 100 - Molecular Genetics 23. Rosalind Franklin and Maurice Wilkins had prepared an X-ray defraction photographof DNA indicating that DNA was long and skinny, helical and consisted of 2 parallelstrandsThe 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 directionsIn each strand each P group is attached to one sugar at the 5’ position (the 5th C) and tothe other at the 3’ position2. The DNA molecule is analogous to the structure of a ladderThe bases on the 2 strands are directed inward and form the rung of the ladder, while thesugar-phosphate face outside and act as the sides of the ladder3. Complimentary base pairing - bases A and T always paired and G and C alwayspaired4. They suggested that the two DNA strands are twisted together to form intertwinnedhelices = double helixThe Structure of RNAThe pentose sugar in RNA is riboseThe pyrimidine thyamine is replaced with uracilThus, the 4 bases are: A, U, C, and GRNA is single stranded, and does not form the double helix like DNAThere are 3 different classes of RNA: mRNA, rRNA, and tRNADNA has 2 primary functions:1. To replicate the stored information it contains2. Use the stored information in the synthesis of proteinsCentral Dogma of molecular genetics:DNA ---------------------------> RNA --------------------> proteintranscriptiontranslation

Bio 100 - Molecular Genetics 3DNA Replication: A Closer LookThe double stranded DNA molecule lends itself to replication because each strand canserve as a template for the formation of a complimentary strandDNA replication requires the following steps:1. The double helix unwinds and unzipsThe unwinding is largely due to the activity of helicaseUnzipping occurs at a distinct site along the chromosome and creates a replication bubbleThe y-shaped junction at the spot where the 2 strands are unwinding is called thereplication fork2. Complimentary Base PairingThe exposed sequence of bases are paired with free nucleotides that drift into the area3. JoiningBase pairs are joined together in a process called polymerizationIt is catalyzed by DNA polymeraseNote: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’ directionDNA replication is continuous on this side of the templateDNA replication is discontinuous on the other templateAn enzyme, ligase, will later link these stretchesIn the end you get 2 double stranded DNA molecules, identical to one another;replication is semiconservativeAccuracy of the ReplicationIn some cases, bases pairs do not pair correctly with one another during replicationNonetheless, the error rate is minimal because DNA polymerase has a "proofreading"functionOn average there is one mistake per billion nucleotides, or 3 mistakes per mitosis

Bio 100 - Molecular Genetics 4Transcription (DNA ----> RNA)During transcription, the DNA code is passed to mRNA1. A segment of the DNA helix unwinds and unzips, and complimentary RNAnucleotides pair with the DNA nucleotides of one strand2. Initiation - RNA nucleotides are joined by the enzyme RNA polymerase, and themRNA molecule is formedRNA polymerase must be must be instructed where to start and where to stop thetranscribing processThe “start transcribing” signal is a nucleotide sequence called a promoter – located inthe DNA next to the beginning of the gene3. After initiation, the RNA (mRNA) strand is elongatesFinally, there is termination – where the RNA polymerase reaches a special sequence ofbases in the DNA template called the terminatorRNA ProcessingIn eukaryotes, after the mRNA strand is formed, it passes from the nucleus to thecytoplasm where it becomes associated with ribosomesBut, before it leaves the nucleus, the mRNA is modified or processedOne kind of processing is the addition of extra nucleotides (The “cap” and “tail” ) to theends of the RNA transcriptAnother type of processing results from the fact that DNA is comprised of long stretchesof noncoding regions (=introns) that interrupt nucleotide sequences that code for aminoacidsThe coding regions - the parts of the gene that are expressed - are called exonsBoth exons and introns are transcribed from DNA into RNARNA splicing - before the RNA leaves the nucleus, the introns are removed and theexons are joined to produce the mRNA with a continuous coding sequence

Bio 100 - Molecular Genetics 5Translation (mRNA -----> protein)During translation the sequence of bases of the mRNA is translated into a sequence ofamino acids3 nucleotide units of the mRNA that codes for a amino acid is called a codone.g., CGU codes for the aa ArginineAlso, there are 3 codons called stop codons, that serve as a signal polypeptide chainterminatione.g., UAA, UAG, UGAOne codon is a start codon that signals to begin polypeptidee.g., AUGTranslation: A Closer LookDuring translation, the sequence of codons in mRNA dictates the order of the aminoacids in the polypeptideTranslation requires the efforts of several enzymes, and 2 other types of RNA: rRNA andtRNArRNAIt makes up the ribosomes; they consist of 2 subunits, each of which is comprised ofrRNA and proteinsrRNA is transcribed from DNA in the area of the nucleolusRibosomes function in coordinating protein synthesisEach ribosome has a binding site for mRNA on its small subunitIts large subunit serves as a binding sites for tRNA: the P site holds the tRNA carryingthe growing polypepetide chain; the A site holds a tRNA carrying the next amino acid tobe added to the chaintRNAtRNA is also transcribed in the nucleus and later moves to the cytoplasmtRNA brings amino acids from the cytoplasm to the ribosomes located in the cytoplasmAmino 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 iscalled the anticodon

Bio 100 - Molecular Genetics 6There are 3 important steps in translation: initiation, elongation, and termination1. InitiationThe mRNA molecule binds (5’ end) to a small ribosomal subunitInitiation begins at the area of the start codon (AUG) of m RNAThe first tRNA pairs with the start codon of mRNAA large ribosomal subunit then attaches to the smaller unitThis completes the ribosomal structureIt possess 2 active sites of attachment for tRNA2. ElongationThe ribosome moves down the length of the mRNA (5' ---> 3')It stops at the second and third mRNA codonsTwo tRNAs are then received by these codons, each carrying a specific amino acidThe 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 andre-enter the cytoplasms tRNA poolWhen each of the tRNAs with an amino acid are attached to the ribosome, the enzymepeptidyl transferase forges a peptide bond between 2 amino acids3. TerminationThe process continues until the ribosome encounters one of three possible stop codonse.g. UAG, UAA, UGAThe is no tRNA to recognize these codons; instead a release factor will bind to the stopcodonThe last tRNA is released and the polypetide chain is freedThe 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