Microbiology Test 3 - Lecture notes Exam 3 PDF

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Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy Microbiology Test 3 Genomes and Chromosomes Chapter Overview: - DNA 101 - Genetic code: BIG picture - Microbial genomes - Mechanism of Gene Transfer - Genome Organization - Plasmids - The features of eukaryotic chromosomes Introduction: - Genetics: the study of genes - Chromosomes: structures containing DNA, the chromosomes contain genes - Genes: segments of DNA that code for functional products, usually proteins - Genome: all the genetic information in a cell Genetic Code: the BIG picture - The genetic code is a set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein

Replication Mutation

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Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy Types of Mutation

Introduction: microbial genomes - Microbial genomes consist of one (usually) or more DNA chromosomes o exception is some viruses have RNA genome - extrachromosomal DNA o Plasmids - Genome: complete set of genetic information referred to as genome DNA: Gene Transfer - Two types of gene transfer are known: o 1. Vertical transmission: from parent to offspring o 2. Horizontal transmission: transfer of small pieces of DNA from one cell to another  DNA-mediated transformation  Transduction

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy  Conjugation Microbial Genetics – DNA - In 1928, Frederick Griffith discovered transformation in bacteria - In 1944, Oswald Avery and colleagues showed that the transforming substance is DNA - In 1953, Rosalind Franklin used X-ray crystallography to determine that DNA is a double helix - Later In 1953, James Watson, Francis crick and Maurice Wilkins discovered the complementary bases and antiparallel nature of DNA Mechanisms of Gene Transfer - DNA-mediated transformation: the transfer of naked DNA from one bacterium to another - Discovered by Frederick Griffith in 1928 while working with Streptococcus Pneumoniae S strain (heat killed)  inject into mice  mice live S strain (heat killed) +  inject into mice  mice die R strain (live) Griffith Experiment - In the 1950’s, conjugation was discovered o Horizontal gene transfer mechanism requiring cell-to-cell contact, which could transfer large segments of some bacterial chromosomes - Allows genes to be mapped relative to one another according to time of transfer o Results suggested that bacterial chromosomes were circular - We know that there is tremendous diversity in prokaryotic genomes Mechanism of Genetic Transformation in Bacteria

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

Genome Organization - prokaryote chromosomes range from 130 to 14,000 kilobase pairs (kb) o Eukaryotic chromosomes can be as big as 100 million kb o Human genome is over 2 million kb - Genomes contain noncoding DNA o > 90% of eukaryotic genomes o Only < 15% of prokaryotic genomes - Prokaryotic genomes includes extrachromosomal DNA (plasmids) - Kb = kilobase pairs = 1000 base pairs Functional Units of Genes - A gene is the basic physical and functional unit of heredity - A gene can operate independently of others - It may exist in tandem with other genes in a unit called an operon - Humans have bw 20,000 and 25,000 genes DNA Structure - DNA, deoxyribonucleic acid, is a polymer of nucleotides - Each nucleotide consists of three parts: o 1. Nitrogenous base  Purine: adenine and guanine

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy  Pyrimidine: cytosine and thymine o 2. Deoxyribose sugar o Phosphate Chemical structure of DNA - Nucleotides are connected to each other by 5’-3’ phosphodiester bonds - Hydrogen bonding allows complementary base interactions - The two backbones of DNA are antiparallel DNA Double Helix - 2 phosphodiester backbones form the double helix - At high temp. (50-90 C) the hydrogen bonds in DNA break and the duplex falls apart, or denatures, into two single strands o High G-C sequences require more energy to denature The Bacteria Nucleoid - Bacteria pack their DNA into a series of loops or domains, collectively called the nucleoid o Loops are anchored by histone-like proteins (HU and H-NS) o E. coli nucleoid contain bw 30-100 loops o Each loop supercoil independent of other loops DNA Supercoiling - There are 2 types of supercoils: o Positive supercoils: DNA is overwound o Negative supercoils: DNA is underwound - Eukaryotes, bacteria, and most archaea possess negatively supercoiled DNA - Some archaeal species living in acid at high temp. have positively supercoiled DNA - Enzymes that change DNA supercoiling are called topoisomerases - Negative supercoils are more loose but still supercoiled Topoisomerases Supercoil DNA - A cell has two types of topoisomerases o Type 1 topoisomerase

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

1) Cleaving both strands at one site in the molecule 2) passing an intact part of the molecule bw ends of the cut site and 3) reconnecting the free ends Plasmids - 2 kinds of extragenomic DNA molecules can interact with bacterial genomes: o Horizontally transferred plasmids o The genomes of bacteriophages (viruses that infect bacterial cells) - Plasmid-encoded functions can contribute to the physiology of the cell o For example, antibiotic resistance Eukaryotic Chromosomes - Eukaryotic genomes are larger than those of bacteria - Because their chromosomes are linear, eukaryotes require a reverse transcriptase called telomerase to replicate their ends - Eukaryotic cells pack their DNA within the nucleus using proteins called histones - A large portion of eukaryotic chromosomes are composed of noncoding DNA: o Introns and pseudogenes Archaeal Genomes - Archaeal genomes combine features of bacteria and eukaryotes o Like bacteria, archaea have:  Polygenic operons  Asexual reproduction  Cells lacking a nuclear membrane  A single circular chromosome o In most species of archaea, however, the processes of DNA replication, transcription, and translation more closely resemble those of eukaryotes - Archaea are not medically important because their optimal living conditions are not similar to human body environment - However, archaea can accept DNA from other organisms which could cause problems in the future

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy Summary - A genome is all the genetic info that defines an organism - The prokaryotic genome is typically a single, circular chromosome, whereas the eukaryotic genome consists of multiple, linear chromosomes - The DNA structure consists of a double helix, composed of four different nucleotides - The bacterial chromosome is packed in a series of protein-bound loops collectively called the nucleoid - Topoisomerases are enzymes that supercoil DNA - Plasmids are autonomously replicating, extra-chromosomal DNA elements o They benefit the host under certain conditions Bacterial DNA Replication Chapter Overview - Overview of bacterial DNA replication - Terminating replication - Plasmid replication DNA Replication - Microbial DNA needs to replicate itself as accurately and as quickly as possible - Bacterial replication involves a number of proteins and genes coming together in a complex machine o E. coli have a doubling time of about 20 minutes o But, it takes 40 minutes just to copy its chromosome o E.coli genome = 4.6 or 4600 depending on unit - Multiple DNA replication can happen at the same time Overview of Bacteria DNA Replication - Replication of cellular DNA is most cases is semiconservative o Daughter cell receives one parental and one newly synthesized strand - Replication is bidirectional o Start at a fixed origin and progress in opposite directions

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

DNA Replication – summary - DNA polymerase III adds nucleotides to the growing DNA strand - In the 5’  3’ direction - Initiated by an RNA primer (DNA primase) - Leading strand is synthesized continuously, - Lagging strand is synthesized discontinuously, creating Okazaki fragments - RNase H removes RNA primers; Okazaki fragments are joined by the DNA polymerase I and DNA ligase Major Proteins involved in DNA replication include: - DnaA: initiator protein - DnaB: helicase, unwinds double helix - DNA primase: synthesis of RNA primer

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy - DNA Pol III: major replication enzyme - DNA gyrase: relieves DNA supercoiling - DNA Pol I: replaces RNA primer with DNA - DNA ligase: joins Okazaki fragments Energy Replication is supplied by necleotides

Terminating Replication - There are as many as ten terminator sequences (ter) on the Escherichia coli chromosome - A protein called Tus (terminus utilization substance) binds to these sequences and acts as a counter-helicase - Ringed catenanes formed at the completion of replication are separated by topoisomerase IV and XerCD proteins Plasmids - Two kinds of extragenomic DNA molecules can interact with bacterial genomes: o Horizontally transferred plasmids o The genomes of bacteriophages (viruses that infect bacterial cells) - Plasmid-encoded functions can contribute to the physiology of the cell o For example, antibiotic resistance Plasmids Replicate Autonomously - Plasmids are much smaller than chromosomes o Found in archaea, bacteria, and eukaryotic microbes

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy o Usually circular o Need host protiens to replicate - Plasmids replicate through rolling circle mechanism

Plasmid Properties - Plasmids have tricks to ensure their inheritance: o Low-copy number plasmids segregate equally to daughter cells o High-copy number plasmids segregate randomly to daughter cells - Plasmids are advantageous under certain conditions: o Resistance to antibiotics and toxic metals o Pathogenesis o Symbiosis - Plasmids can also be transferred between cells Chapter Summary -

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DNA replication is divided into three phases: o 1. Initiation: occurs at the origin (oriC) o 2. Elongation: occurs at the replication forks o 3. Termination: occurs at the terminus (ter) Each phase requires a number of different proteins Plasmids are autonomously replicating, extra-chromosomal DNA elements o They benefit the host under certain conditions

Transcription and Translation Chapter Overview - RNA polymerases and sigma factors - Transcription: DNA is converted to RNA - The genetic code, ribosomes, and tRNAs - Translation: RNA is converted to protein - How proteins are modified and folded - How proteins are secreted

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy - How proteins are degraded RNA Structure - RNA, ribonucleic acid, differes from DNA: o Usually single-stranded  RNA-RNA double strand called hairpins o Contains ribose sugar o Uracil replaces thymine Different Classes of RNA - There are several classes of RNA, each designed for a different purpose o Messenger RNA (mRNA): encodes proteins o Ribosomal RNA (rRNA): shuttles part of ribosomes o Transfer RNA (tRNA): shuttles amino acids o Small RNA (sRNA): regulates transcription or translation o tmRNA: frees ribosomes stuck on damaged mRNA o Catalytic RNA: carries out enzymatic reactions - Some RNA act as scaffolds to build/transport or act as an enzyme and facilitate the chemical reaction The Central Dogma - The cell accesses its vast store of data in its genome by: o Reading a DNA template to make an RNA copy (transcription) o Decoding the RNA to assemble protein (translation) - the chromosome is huge compared to RNA so in this process small copies are made of DNA in form of RNA to make multiple copies of protein - every time we unwind double stranded DNA it is exposed to enzymatic reaction - DNA is a master copy while RNA is a zerox copy - Every time that info is transferred form RNA to protein the RNA has to be recycled

Functional Units of Genes - A gene is the basic physical and functional unit of heredity - A gene can operate independently of others - It may exist in tandem with other genes in a unit called an operon Transcription: BIG picture

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

- Coding strand – same as info on transcribed DNA however T becomes U - Know how to relate RNA to double stranded DNA - Nucleotide can only be added to 3’ end on RNA Transcription - Transcription in the synthesis of a strand of RNA from a DNA template by RNA polymerase (RNA pol) - Begins when RNA pol binds to the promoter on DNA o Promoter is a sequence of DNA upstream of the transcription start site (+) RNA Polymerases and Sigma Factor - RNA pol is a complex enzyme that carries out transcription by making RNA copies (transcripts) of a DNA template strand - in bacteria, the RNA pol is composed of:

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy o sigma factor  required for the initiation phase o core polymerase: alpha 2, beta, beta prime  required for the elongation phase - the sigma factor helps the core enzyme detect the promoter, which signals the beginning of the gene - every cell has a “housekeeping” sigma factor - a single bacterial species can make several different sigma factors - E. coli have predominantly sigma-70 (sigma 70) o Recognizes consensus sequences at the -10 and -35 positions, upsteam of start of the RNA transcript (+1) - What happens if cell stops making sigma factor? o There will be no transcription - Sigma – helps housekeeping genes (have to be transcribed throughout the life of the cell) the cell needs this all the time (histones) Transcription of DNA to RNA - Transcription occurs in three phases: o 1. Initiation: RNA pol binds to the promoter  This is followed by melting of the helix and synthesis of the first nucleotide of the RNA  Initiated by sigma factor o 2. Elongation: the RNA chain is extended o 3. Termination: RNA pol detaches from the DNA, after the transcript is made 1. Initiation of transcription - RNA pol, by the help of sigma factor, bind to the promotor region - Results in unwinding of one helical turn - RNA polymerase then starts transcription o the first nucleotide of the new RNA chain is usually a purine (A and G) o Sigma factor discarded after the first few RNA bases are made 2. Elongation of RNA transcripts - RNA pol move along the template, synthesizing RNA at ~45 bases/sec - DNA unwind ahead forming a 17-bp transcription bubble - DNA unwinding produces positive supercoils ahead, which are removed by DNA topoisomerases o Which topoisomerases? - RNA polymerase creates the phosphoester bond 3. Termination of transcription - All bacterial genes use one of two known transcription termination signals: o 1. Rho-dependent  Relies on a protein called Rho  Contact between Rho and RNA pol cause termination o Rho-independent

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy  Relies on a NusA protein  Contact bw NusA and RNA pol cause termination o what determines when transcription is going to end?  translation Different Classes of RNA - there are several classes of RNA, each designed for a different purpose: o messenger RNA (mRNA): encodes proteins o ribosomal RNA (rRNA): integral part of ribosomes o transfer RNA (tRNA): shuttles amino acids o small RNA (sRNA): regulation transcripts or translation o tmRNA: frees ribosomes stuck on damaged mRNA o catalytic RNA carries out enzymatic reactions Antibiotics that Affect Transcription - antibiotics must meet two fundamental criteria: o they must kill or retard the growth of a pathogen, and they must not harm the host - Rifamycin B o Selectively binds to the bacterial RNA pol o Inhibits transcription initiation - Actinomycin D o Nonselectively binds to DNA o Inhibits transcription elongation Translation of RNA to protein - Message in mRNA are arranged in a sentence of triplets of nucleotides, called codons, represent individual amino acids - Ribosomes are the machines that read the language of mRNA and translate it into protein o They do so via the genetic code o And tRNA decodor molecules that convert the language of RNA into that of proteins The Genetic Code - There are 64 possible codons: o 61 specify amino acids  Include the start codons o 3 are stop codons (UAA, UAG and UGA) - The code is degenerate or redundant o Multiple codons can encode the same amino acid o E.g. GCU, GCC, GCA, GCG code for Alanine tRNA - tRNA has clover leaf shape - a tRNA molecule has 2 functional regions: o anticodon: hydrogen bonds with the mRNA codon specifying an amino aicd o 3’ (acceptor) end: binds the amino acid

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy - tRNAs contain a large number of unusual, modified bases

Codon-anticodon pairing - the tRNA anticodon consists of three nucleotides - the anticodon hydrogen bonds with the mRNA codon in an antiparallel fashion - tRNA is “charged” with an amino acid covalently attached to the 3’ end - cell has generally 20 of these “match and attach” tRNA, one for each amino acid

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy

The Ribosome, a Translation Machine - ribosomes are composed of 2 subnunits, each of which includes rRNA and proteins - in prokaryotes, the subunits are 30S and 50S and combine to form the 70S ribosome o S, Svedberg unit, is a measure of rate of sedimentation in centrifugation rather than size

16S rRNA Gene Sequences - The 30S subunit has a 16S subunit (1540 nucleotides) - 16S rRNA gene is used in reconstructing phylogenies - while highly conserved, there are differences in 16S rRNA sequences that increase in relation to the evolutionary distance among species - 16S rRNA serves as a molecular clock How do Ribosomes Find the Right Reading Frame? - Every mRNA has three potential reading frames, so how does the ribosome find the right one? o The upstream, untranslated leader RNA contains a purine-rich sequence with the consensus 5’-AGGAGGU-3’

Know antibodies and how they work Bacteriacytol – anything that attacts the cell wall (kill bacteria) Bacteriastataic – stops bacterial growth but does not destroy  Located 4-8 bases upstream of the start codon in E. coli o This Shine-Dalgarno sequence is complementary to a sequence at the 3’ end of 16S subunit The Three Stages of Protein Synthesis - P...