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Microbial Genetics • Slides are adapted from Dr. Gary Andersen, Curtis Smith, KCKCC • Reference: Chapter 7,8 from (Black, J., 2005) Basic Units of Genetics • Genomes – the total of the genetic material in a cell. • Gene - The unit of heredity for a given genetic trait. The site on a DNA molecule that carries the code for a certain cell function. • Viruses – 4 or 5 genes, E. coli – 4228 genes, Human ~ 31,000 genes. Nucleic Acids I. Nucleic acids are located in the nucleoid of bacteria, and the nucleus of eukaryotes. There are 2 kinds of nucleic acids: RNA & DNA. Ruptured E. coli cell showing DNA DNA A. CHARACTERISTICS OF DNA DNA(deoxyribonucleic acid) is made of subunits called nucleotides. Nucleotides are made of 3 components. These 3 components are linked together with a covalent bond. E. Coli = 4.6 million nucleotide pairs (~1mm) Corn = 2.5 billion nucleotide pairs Human = 3 billion nucleotide pairs (2nm wide by 2 meters long) Significance of DNA Structure • Maintains the code with high degree of fidelity. (double strand assures accurate replication) • Provides a method for introducing a high degree of variety. (unlimited variety of sequences possible) 1. COMPONENT 1 - Phosphate Phosphate group - Phosphate functions as a structural part of nucleic acids. 2. COMPONENT – Ribose Sugar 2, DEOXYRIBONUCLEIC ACID Ribose - A five carbon sugar that functions as part of the DNA backbone (ie. structural). “2, Deoxy” means without oxygen on the number 2 carbon atom. 3. COMPONENT – Nitrogen Bases NITROGEN CONTAINING BASES Function: express genetic information. Composition : 2 PURINES: ADENINE (A) double ring structures GUANINE (G) 2 PYRIMIDINES: THYMINE(T) CYTOSINE(C) single ring structures Nucleotide Base Composed of one Nitrogen base, one Deoxyribose, and one Phosphate group Deoxyribose Phosphate Adenine (Nitrogen base) • 4 Nucleotides D N A S t r u c t u r e • DNA Structure 4. DNA is a double helix (there are 2 strands of DNA) which are intertwined with 5 base pairs per turn. 5. DNA has complimentarity that is A always bonds with T and G always with C 6. DNA is always antiparallel. The 2 strands of DNA are always oriented in opposite directions. ( 5’ PO3 end – 3’ OH end) http://www.umass.edu/microbio/chime/dna/dna53.htm DNA Bonds • 3-D Image of DNA B. RNA RIBONUCLEIC ACID Similar to DNA except: 1. RNA is single stranded 2. RNA has a ribose sugar instead of deoxyribose. (Oxygen on #2 C). 3. RNA has URACIL (u) instead of thymine 4. RNA is always shorter than DNA, ~ 1,000 nucleotides in length • C. FUNCTIONS OF RNA 1. rRNA (ribosomal) - comprises the ribosome (site of protein synthesis). (60% of a ribosome is made of RNA, the rest is protein). 2. tRNA (transfer) carries amino acids to the ribosome during protein synthesis. Also known as the “ANTICODON” 3. mRNA (messenger) - a complimentary strand of RNA equal in size to 1 gene (normally ~1,000 nucleotides). “CODON” - coded info from DNA (bound for the ribosome) THE CENTRAL DOGMA OF BIOLOGY “Francis Crick – 1956” There are 3 parts to the flow of information in all cells. Transcription Translation DNA -------------mRNA-----protein Replication Central Dogma of Biology DNA REPLICATION 1. Where 2 parental strands of DNA are copied into 2 daughter strands. Rate = 1,000 nucs per seconds without error. This leads to binary fission in bacteria. Cell Division) = 2 daughter cells 2. Each cell receives 1 parental strand and 1 daughter strand. (semiconservative replication) GA sp 08 As the two replication forks meet, the two new chromosomes separate—each containing one new and one old strand Replication bonding R e p l i c a t i o n F o r k • 1. EVENTS IN DNA REPLICATION a. DNA unwinds using the enzyme DNA Helicase b. SSBP holds the 2 strands apart (single strand binding proteins) c. Note: 2 replication forks. DNA replication is considered bi-directional replication. DNA REPLICTION CONT’D d. Polymerization requires DNA Polymerase (POL III) which is an enzyme that synthesizes 2 nucleotide strands (daughter strands) from 2 parental (templates) strands. e. DNA exonuclease (POL I) removes any mistaken base pairs. f. DNA ligase seals any gaps and joins the 2 strands together. DNA Replication Enzymes at Work • Steps in Replication • Replication of DNA cont’d Sit back!! We’re watching a movie for a while (But don’t expect too much) Let’s watch some “interesting”, academic movies • Movie 1 • Movie 2 • Movie 3 THE CENTRAL DOGMA OF BIOLOGY There are 3 parts to the flow of information in all cells. Transcription Translation DNA --------------mRNA---------protein Replication B. TRANSCRIPTION 1. 2nd part of the central dogma of biology 2. 1st step in gene expression (i.e.protein synthesis). 3. The cells genetic plan contained in DNA is transcribed into a complimentary base sequence called messenger RNA (mRNA). 4. The region of DNA that produces or serves as a template for mRNA is called a gene. A gene normally consists of around 1,000 base pairs. It is the smallest segment of DNA that codes for mRNA. TRANSCRIPTION CONT’D 5. RNA polymerase is the enzyme responsible for making mRNA Transcription continued 7. Example: DNA DNA mRNA A T G C C G T A C G G C A UG C C G 8. mRNA is a blueprint of DNA or a transcript or code. 9. One code word consists of three letters. Animation of Transcription • Movie 1 • Movie 2 • Movie 3 C. TRANSLATION 1. Translation is the 3rd part of the central dogma of biology (2nd step in gene expression or protein synthesis). 2. After transcription, the coded information in mRNA is translated into an enzyme (protein). 3. This process takes place on the ribosome. Note that the ribosome is made of rRNA and protein. Translation Graphic TRANSLATION CONT’D 4. tRNA STRUCTURE tRNA utilizes the information in mRNA to determine the sequence of amino acids in a protein. tRNA has a cloverleaf shape. The amino acid end binds one specific amino acid in the cytoplasm. The anticodon end pairs with the codon on mRNA. Transfer RNA Structure TRANSLATION CONT’D 3. The mechanics of translation Initiation; mRNA bumps into the small subunit and triggers the two ribosomal subunits to bind together. The first tRNA anticodon (UAC) carrying the amino acid methionine hydrogen bonds with the codon AUG on mRNA. TRANSLATION CONT’D b. Elongation – The second tRNA binds to the second code word on mRNA. A peptide bond forms between the two amino acids. The first tRNA leaves, and the enzyme translocase moves the ribosome down one code word of mRNA at a time. This repeats ~ 300X. TRANSLATION CONT’D In termination, one of three possible stop codons is reached. The last tRNA falls away and the two ribosomal subunits fall apart. C. d. The Genetic Code 61 sense codons for 20 amino acids 3 nonsense (or stop codons) 64total codons Pg 180 (Black, J., 2005) The Genetic Code • Steps in Protein Synthesis Steps in Protein Synthesis Steps in Protein Synthesis Steps in Protein Synthesis Steps in Protein Synthesis Protein Synthesis Animation time.. • Movie 1 • Movie 1 Translation - Animation