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Chapter 13 - Gene Function What do our genes actually encode for? PROTEINS How do we make proteins from our genes? Transcription – synthesis of RNA from a DNA template Translation – synthesis of protein from a RNA template Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Transcription Why do we need to make RNA from DNA first? Believe it or not it’s a great regulatory site If we don’t need a particular protein, do our cells want to waste making it? How does a cell know when to express a particular gene (DNA sequence) & how does the cell control this expression? The ability to form an intermediate coding molecule (RNA) is the answer to these questions & a simple example of this control is depicted by the bacterial lac operon Chapter 13 - Gene Function Chapter 13 - Gene Function Structure RNA 1. Ribose – 5 carbon sugar – forms part of the backbone structure 2. Nitrogenous base – Information is coded in the sequence of these bases A. Purines – Double ring structure – Adenine (A) & Guanine (G) B. Pyrimidines – Single ring structure – Uracil (U) & Cytosine (C) 3. Phosphate group – forms part of the backbone structure RNA is single-stranded Chapter 13 - Gene Function Chapter 13 - Gene Function Types of RNA Ribosomal RNA (rRNA) – associates with proteins to form the ribosome, the structural site for protein synthesis (protein synthesizing factory) Transfer RNA (tRNA) – responsible for connecting & carrying amino acids to ribosome for synthesis into a protein Messenger RNA (mRNA) – the template for translation (protein synthesis) – a sequence of nucleotides that will determine the sequence of amino acids in the final protein – it is a copy of the DNA BLUEPRINT! Chapter 13 - Gene Function Chapter 13 - Gene Function Transcription Transcription factors regulate the initiation of transcription They along with RNA polymerase are attracted to a sequence of DNA known as the promoter The DNA unwinds via the mechanism already discussed & RNA polymerase facilitates the growth of the RNA molecule 1. No primer is needed, unlike DNA replication 2. Only one strand of the DNA will serve as the template 3. RNA transcription is directional: 5’ to 3’ 4. Multiple RNA strands can be simultaneously transcribed Chapter 13 - Gene Function mRNA processing Before the mRNA can be used for translation it must be processed into a final coding sequence. It will undergo the following: 1. Capping – Short sequences of nucleotides added to the 5’ end 2. Tailing – Addition of adenine nucleotides to the 3’ end 3. Splicing – Removal of introns (non-coding sequences) & the joining of exons (coding sequences) Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Translation How do we make a protein from RNA? What do we need? 1. Strand of mRNA 2. Ribosomes: large & small subunits 3. Charged tRNAs – tRNAs with an attached amino acid Translation is divided into 3 stages 1. Initiation 2. Elongation 3. Termination Chapter 13 - Gene Function How does the mRNA serve as the template for translation? How is the correct sequence of amino acids obtained? The mRNA is divided into 3 nucleotide sections called codons Each codon encodes for an amino acid as depicted on the following table Chapter 13 - Gene Function Chapter 13 - Gene Function How does tRNA transfer amino acids to a growing polypeptide chain? tRNAs link up to the codons on the mRNA via their own anticodons – a three nucleotide sequence that is complementary with the codon’s sequence Each tRNA molecule carries a specific amino acid, so that when the anticodon & codon match up within the ribosome, the amino acids are assembled in the proper sequence Chapter 13 - Gene Function Thus if we have a sequence of the following codons: 5’ – CCC – CAC – UUU – AAU –UAG 3” What is the sequence of amino acids that is encoded for by this mRNA? Proline – Histidine – Phenylalanine – Asparagine - Stop Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function Translation specifics Translation proceeds in a 5’ to 3’ direction on the mRNA The codon is a 3 nucleotide sequence on the mRNA For each codon there exists an anticodon on a tRNA The tRNA is responsible for carrying a specific amino acid to its codon on the mRNA Peptide bonds are formed between the amino acids by enzymes located in the ribosomal complex Multiple proteins can be translated simultaneously like RNA transcription polyribosome Chapter 13 - Gene Function Mutations How do mutations in our DNA lead to phenotypic changes? Two categories of mutations Point mutations – change in a single DNA base which leads to: 1. Missense mutation – change in amino acid due to the DNA base change 2. Nonsense mutation – DNA base change causes the appearance of a “stop” codon 3. Silent mutation – DNA base change does not change the amino acid sequence Insertion / Deletion mutations – adding or removing DNA bases which leads to a: 1. Frameshift mutation – a disruption in the reading frame which can lead to changes in all amino acids downstream of the insertion or deletion Chapter 13 - Gene Function Chapter 13 - Gene Function Quick little example Normal DNA THE ONE BIG FLY HAD ONE RED EYE Missense THE ONE DIG FLY HAD ONE RED EYE Nonsense THE ONE BIG FLY Frameshift THE ONE DBI GFL YHA DON ERE DEY Chapter 13 - Gene Function Chapter 13 - Gene Function Chapter 13 - Gene Function PRACTICE QUESTIONS 1. What enzymes are used during the steps of transcription? Translation? 2. What are the 3 types of RNA? 3. Define the following terms: codon, anticodon, semi-conservative replication, polyribosome 4. What direction does replication, transcription, & translation occur? 5. Given that a template strand of DNA is 3’ TAC AAA ACC CCG GAG ATC 5’, transcribe the appropriate mRNA (assuming no introns) & translate the appropriate protein. 6. What is the difference between a nonsense, missense, & silent mutation? 7. What is a polyribosome? How is this structure similar to the transcriptional structure? 8. What is an exon? Intron?