BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus Chapter 9- An Introduction to Microbial Genetics* *Lecture notes are to be used as a study guide only and do not represent the comprehensive information you will need to know for the exams. 9.1 Introduction to Genetics and Genes: Unlocking the Secrets of Heredity Genetics is the study of heredity, passing genetic information from parent to offspring. The study of genetics can take place at many levels: organism, cell, chromosome, molecular (figure 9.1). The study of microbial genetics can be applied to universal themes, and serves as the basis for genetic engineering in chapter 10. The Nature of the Genetic Material Deoxyribonucleic acid, or DNA is the molecule of genetics. It is important to understand its structure, organization and function. The Levels of Structure and Function of the Genome As you read the sequence of nucleotides in a molecule of DNA (e.g. ATGCCCTAA...etc.), there are regions, specific sequences with beginnings and ends, which define genes. These sequences are the regions that encode the information for building proteins. Another way of saying this is that a gene is a sequence of nucleotides that codes for RNA and in most cases ultimately for the synthesis of a protein. An organism’s genome is the sum of all its genetic material (DNA). The term chromosome refers to a discrete DNA molecule; whereas prokaryotes tend to have only one, eukaryotes can have many (in humans there are 23 unique, individual chromosomes). Structural genes code for proteins; other genes code for tRNAs and rRNAs. Also found in bacteria are additional pieces of extra-chromosomal DNA called plasmids. These small (~500- 3000 nucleotide base pairs), circular pieces of DNA can comprise as much as 10% of the total genetic information in a bacterium. Resistance plasmids (R plasmids or R factors) are so-called since they give bacteria resistance (i.e. immunity) to a variety of antimicrobial drugs1 (antibiotic resistance). Other functions plasmids encode include: virulence factors (the ability to cause disease), heavy metal resistance, and resistance to bacteriophage attack. Bacteria can not only transfer plasmids between cells of the same species, but they can also transfer DNA between genera: Shigella, Salmonella, Escherichia, Yersinia, Klebsiella, Serratia, and Proteus can all transfer plasmids. Transfer of antibiotic resistance genes is a growing problem for therapeutics. Note: Therapeutics Throughout the following sections, take note of the fact that many pharmaceutical agents used to treat infections target DNA replication, transcription, and translation. Treatment of bacterial infections with such drugs is based on the premise that the growth of the pathogen will be inhibited by blocking its protein 1 Antibiotic resistance is covered in detail in chapter 12. 1 BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus synthesizing machinery selectively, without disrupting the cellular biochemistry of the patient receiving the therapy. The genetic makeup of an organism is called its genotype, and when the gene is expressed it produces a phenotype, a characteristic you can see. The Size and Packaging of Genomes The bacterial chromosome varies in size from fewer than 1,000,000 (106) nucleotide base pairs that code for about 1000 proteins (mycoplasmas) to 4.5 x 106 nucleotide base pairs (E. coli), that code for about 4,500 proteins. (Humans = 3 x 109 nucleotides and approximately 25,000 genes). The Packaging of DNA The circular chromosomal DNA of bacteria is packaged by a topoisomerase enzyme, specifically called DNA gyrase. The Structure of DNA: A Double Helix with Its Own Language Nucleic acids are large organic molecules found in all cells. There are 2 types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are large polymers of subunits called nucleotides. Each nucleotide contains a 5-carbon sugar (either deoxyribose or ribose), a phosphate group, and one of 5 different nitrogen-containing molecules called nitrogenous bases. The sugar and the phosphate form a backbone and the nitrogen bases stick out as side branches. P S-N P S-N fig. 2.24 ; fig. 2.25; fig. 9.B P S = sugar, P = phosphate, N = nitrogenous base S-N P S-N The nitrogenous bases can be categorized as purines and pyrimidines (fig. 2.25). Purines (2 rings): adenine and guanine (A and G) Pyrimidines (1 ring): thymine, cytosine and uracil (T, C, and U) They are abbreviated using a capitalized first letter - A, G, T, C, and U. Adenine, guanine, thymine and cytosine are found in DNA (fig 2.24). Adenine, guanine, cytosine and uracil are found in RNA. The sugar in DNA is 2 BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus deoxyribose and the sugar in RNA is ribose (fig. 2.24c). Normally2, DNA is a double-stranded helix while RNA is single-stranded. DNA molecules occur as a double-stranded helix in bacteria, protizoans, plants, fungi, and animals. It has a sugar-phosphate backbone with the purines and pyrimidines covalently linked to the deoxyribose sugar. Purines and pyrimidines are complementary to each other in a very specific fashion. As two separate strands of DNA move closer together, the nitrogenous bases can pair up and be held together by hydrogen bonds: A=T and GC. The base pairing of A with T and G with C is always conserved. Hydrogen bonds are easily formed and easily broken (fig. 2.26; fig. 2.27; fig. 9.4). This allows the helix to “unzip” and each strand can be used to produce a new strand through a process called DNA replication: The physical characteristics of all organisms is determined by the genetic information contained in their cells. Information encoded in sections of the DNA called genes is used by the cell to manufacture the proteins necessary for its survival. This flow of genetic information in cells follows the sequence (figs. 9.8; fig.9.9): (i) (ii) DNA mRNA Protein And is made up of two separate events: (i) Transcription is the process whereby DNA → mRNA (ii) Translation is the process whereby mRNA → Protein DNA Replication: Preserving the Code and Passing it On Bacteria grow through binary fission. Prior to cell division, the bacterium must replicate its DNA. Replication of the chromosome starts at a site called the origin of replication. An enzyme called DNA gyrase releases chromosome supercoils (“unwinds DNA”). After the DNA is unwound, DNA helicase breaks the hydrogen bonds between nucleotides, allowing for it to “unzip” the DNA double helix, thus separating the two strands. The copying enzyme, DNA polymerase, moves in to synthesize new strands based on the open template. Replication proceeds in two directions around the circumference of the chromosome. Occasionally, incorrect bases are paired which can lead to mutations (see below for more info). (see Table 9.1 for enzyme functions.) DNA DNA polymerase 2DNA 2 Exceptions occur in viruses where double-stranded DNA, double-stranded RNA, single-stranded RNA, and single-stranded DNA are found. 3 BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus Replication of DNA A T=A T= T =A T=A T=A A=T A= =T A=T A=T CG C G CG CG T=A T= =A T=A T=A GC G C GC GC A=T A=T A=T A=T T=A T=A T=A T=A GC GC GC GC fig. 2.26; GC GC GC GC fig. 9.5; CG CG CG CG fig. 9.6 1. DNA Gyrase relaxes the supercoiled DNA (unwinding). 2. DNA Helicase breaks the hydrogen bonds between bases (unzips the double helix). 3. DNA Polymerase uses complementary base pairs (G to C, C to G, T to A, and A to T) to construct a new strand. 4. Two helices are produced, each containing one old and one new strand. Therapeutics note: Fluoroquinolones (e.g. Ciprofloxacin) – class of drugs that act by inhibiting bacterial DNA gyrase, thus preventing normal DNA synthesis. 9.2 Applications of the DNA Code: Transcription and Translation Transcription is the process of producing RNA from a DNA template; and translation is the process of producing a protein from a mRNA. The Gene-Protein Connection The Triplet Code and the Relationship to Proteins When DNA is transcribed to mRNA the bases in mRNA are read in triplet, called a codon. Each codon codes for an amino acid (figure 9.9). The Major Participants in Transcription and Translation While the role for DNA is information storage, RNA must perform different jobs inside the cell. There are three primary types of RNA, each with a different function (Table 9.2): 1. Messenger RNA (mRNA) is a single stranded copy of the sequence information in a gene. The sequence of nucleotides in an mRNA is used as a template to make a protein during translation (fig. 9.10). 2. Transfer RNA (tRNA) carries the amino acids to the ribosome (fig. 9.10). tRNAs are short, folded molecules with two distinct ends: one end of the tRNA attaches to a specific amino acid; the other end has an anticodon, or group of 3 nitrogenous bases that are complementary to a codon on mRNAs. 4 BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus 3. Ribosomal RNA (rRNA) is a physical part of a functional ribosome. While rRNA is rarely drawn out in diagrams, consider it to be a necessary subunit of a ribosome and that it acts as an enzyme, forming peptide bonds between amino acids. Summary of differences between DNA & RNA: DNA RNA Nitrogenous bases A, T, G, C A, U, G, C Pentose sugar Deoxyribose sugar Ribose sugar Structure Double Stranded Helix Single stranded Transcription: The First Stage of Gene Expression Transcription refers to the formation of RNA using the DNA sequence as its template.