Microbiology: Module 9 - An Introduction to Microbial Genetics

Chromosomes

- Discrete cellular structure composed of a neatly packaged DNA molecule - Eukaryotic chromosomes are located in the nucleus and are multiple and linear - Bacterial chromosomes are a single circular loop - All chromosomes contain a series of basic informational "packets" called genes* - DNA is double-stranded - Genetic material in Bacteria is double-stranded DNA* Gene (multiple meanings) - Classical Genetics: Fundamental unit of heredity responsible for given trait in an organism - Molecular/Biochemical sense - Portion of the chromosomes that provides info for a given cell function - Specific segment of DNA that contains the necessary information to make a molecule of protein or RNA

Stages from beginning to end of the Transcription process:

1) RNA polymerase binds to the promoter region on the DNA strand. 2) RNA polymerase builds the mRNA strand based on their DNA sequence. 3) RNA polymerase dissociates from the DNA strand at a specific sequence.

Key Points that connect DNA and Protein Function

1. DNA is the blueprint that indicated which kinds of proteins to make and how to make them; this blueprint exists in the order of triplets along the DNA strands 2. The order of triplets directs a protein's primary structure - the order and type of amino acids in the chain - which determines its characteristic shape and function 3. Proteins contribute significantly to the phenotype by functioning as enzymes and structural molecules

Major Events in Transcription

1. Each gene contains a specific promoter region and a leader sequence for guiding the beginning of transcription. Next is the region of the gene that codes for a polypeptide and ends with a series of terminal sequences that stop translation. 2. DNA is unwound at the promoter by RNA polymerase. Only one strand of DNA, called the template strand, supplies the codes to be transcribed by RNA polymerase. This strand runs in the 3' to 5' direction. 3. The RNA polymerase moves along the DNA strand, adding complementary nucleotides as dictated by the DNA template. The mRNA strands reads in the 5' to 3' direction. 4. The polymerase continues transcribing until it reaches a termination site, and the mRNA transcript is released to be translated. Note that the section of the transcribed DNA is rewound into its original configuration.

The Steps of Translation.

1. Entrance of tRNAs 1 and 2. 2. Formation of peptide bond. 3. Discharge of tRNA 1 at the E site. 4. First translocation, tRNA 2 shifts into P site, entrance of tRNA 3. 5. Formation of peptide bond. 6. Discharge of tRNA 2, second translocation, enter tRNA 4. 7. Formation of peptide bond. 8. Process repeated until stop codon is reached.

The Significance of DNA Structure

1. Maintenance of code during reproduction - Constancy of base pairing guarantees that the code will be retained. When strands are separated, each strand serves as a template for replication of the molecule into an exact copy. 2. Providing variety - order of bases responsible for RNA and protein synthesis, thus for the phenotype of each organism.

Stages of DNA replication in order from first to last in prokaryotic cells:

1. uncoiling of the parent DNA molecule 2. unzipping the hydrogen bonds between the base pairs 3. synthesis of two new DNA strands 4. two DNA molecules, each with one old and one new strand

Selected Mutagenic Agents and Their Effects

Agent - Effect: Chemical - Nitrous acid, bisulfite - Remove an amino group from some nitrogen bases Ethidium bromide - Inserts between the paired bases Acridine dyes - Cause frameshifts due to insertion between base pairs Nitrogen base analogs - Compete with natural bases for sites on replicating DNA Radiation - Ionizing (gamma rays, X rays) - Form free radicals that cause single or double breaks in DNA Ultraviolet - Causes cross-links between adjacent pyrimidines

Transduction

Bacteriophage serves as a carrier of DNA from a donor cell to a recipient cell • Two types: - Generalized transduction** - random fragments of disintegrating host DNA are picked up by the phage during assembly; any gene can be transmitted this way - Specialized transduction** - a highly specific part of the host genome is regularly incorporated into the virus

Enzymes Involved in DNA Replication

Enzyme - Function: Helicase - Unzipping the DNA helix Primase - Synthesizing an RNA primer DNA polymerase III - Adding bases to new DNA chain; proofreading the chain for mistakes DNA polymerase I - Removing RNA primers, replacing gaps between Okazaki fragments with correct nucleotides, repairing mismatched bases Ligase - Final binding of nicks in DNA during synthesis and repair Gyrase - Supercoiling • Critical requirement of DNA replication is that each completed daughter molecule be identical to the parent in composition but neither is completely new • Strand that services as a template is an original parental DNA strand and is retained in the daughter molecule • Preservation of the parent molecule in this way, termed Semiconservative Replication*

Genetic transfer of ______ to a methicillin-resistant Staphylococcus aureus (MRSA) strain produces vancomycin resistant enterococci (VRE).

a transposon containing the vanA operon Vancomycin-resistant enterococci (VRE) arise when, through genetic transfer, a MRSA strain acquires the vanA operon located within a transposo.

Classification of Major Types of Mutations

Example: Wild-type (nonmutated) sequence - sequence of a gene is the DNA sequence found in most organisms, generally considered the "normal" sequence *Categories of Mutations based on type of DNA alteration* • Substitution mutations 1. Missense: THE BIG MAD CAT ATE THE FAT RED BUG - A missense mutation causes a different amino acid to be incorporated into a protein. - Effects range from unnoticeable to severe, based on how the new amino acids alters protein function. 2. Nonsense: THE BIG BAD XXX (stop) - A nonsense mutation converts a codon to a stop codon, resulting in premature termination of protein synthesis. - Effects of this type of mutation are almost always severe. • Inversion mutations THE BIG ABD CAT ATE THE FAT RED BUG THE BIBGAD CAT ATE THE FAT RED BUG - Inversion arise when adjacent letters exchange places, which alters 1 or 2 bases, depending on the location of the inversion. - This does not change the reading frame, but can result in significant changes in amino acids and protein function. • Frameshift Mutations 1. Insertion: THE BIG BAB DCA TAT ETH EFA TRE DBU G > 2. Deletion: THE BIG * ADC ATA TET HEF ATR EDB UG > - Insertion (addition of letter) and deletion (removal of letter) mutations cause a change in the reading frame <> of the mRNA, resulting in a protein in which every amino acid after the mutation can be affected. - Because of this, frameshift mutations almost always result in a nonfunctional protein.

Microbial Genomes

Genome** - sum total of genetic material (DNA) in a cell • Most exists as chromosomes • Some appear in non-chromosomal sites: - Mitochondria and Chloroplasts of eukaryotes are equipped w/ their own functional chromosomes - Plasmids - bacteria and some fungi contain these tiny extra pieces of DNA • Genome of cells - only DNA • Genome of viruses - DNA or RNA

Events in DNA Replication - Enzymes

Making an exact duplicate of the DNA involves 30 different enzymes: • Helicase unwinds and unzips the DNA double helix • An RNA primer is synthesized at the origin of replication by a primase • DNA polymerase III then adds nucleotides in a 5′ to 3′ direction: - Leading strand - synthesized continuously in 5′ to 3′ direction - Lagging strand - synthesized 5′ to 3′ in short segments; overall direction is 3′ to 5′ • DNA polymerase I removes the RNA primers and replaces them with DNA • When replication forks meet, ligases link the DNA fragments along the lagging strand • As replication proceeds, one newly synthesized strand loops down • When the forks have gone full circle, a termination site shuts replication down • The two circular daughter molecules remain connected briefly but are nicked and separated by a helicase

Types of Genetic Recombination in Bacteria

Mode - Factors Involved - Direct of Indirect* - Examples of Genes Transferred: Conjugation - Donor cell with pilus; Fertility plasmid in donor; Both donor and recipient alive; Bridge forms between cells to transfer DNA - Direct - Drug resistance; resistance to metals toxin production; enzymes; adherence molecules; degradation of toxic substance; uptake of iron Transformation - Free donor DNA (fragment or plasmid); Live, competent recipient cell, donor usually dead - Indirect - Polysaccharide capsule; metabolic enzymes; drug resistance; unlimited with cloning techniques Transduction - Donor is lysed bacterial cell; Defective bacteriophage is carrier of donor DNA; Live recipient cell of same species as donor - Indirect - Exotoxins; enzymes for sugar fermentation; drug resistance

Causes of Mutations: Spontaneous or Induced

Mutation can be Spontaneous or Induced • Spontaneous mutations - random change in the DNA arising from errors in replication that occur without known cause • Induced mutations - result from exposure to known Mutagens* - physical (primarily radiation) or chemical agents that damage DNA and interfere with its functioning

Please choose all of the statements that are true regarding transcription and translation in prokaryotes.

Prokaryotic mRNAs often contain information from several genes in a series Transcription and translation can occur at the same time in prokaryotes Translation occurs in the cytoplasm of prokaryotes

RNAs: Tools in the Cell's Assembly Line

RNA - an encoded molecule like DNA 1. RNA is a single-stranded molecule that can assume secondary and tertiary levels of complexity, leading to specialized forms of RNA (mRNA, tRNA, and rRNA) 2. RNA contains uracil (U), no thymine (T) like DNA, as the complementary base-pairing mate for adenine (A) 3. The sugar in RNA is ribose rather than deoxyribose

Transposons

Special DNA segments that have the capability of moving from one location in the genome to another - "jumping genes" • Cause rearrangement of the genetic material • Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome • May be beneficial or harmful • Overall effect of transposons - to scramble the genetic language - can be beneficial or adverse, depending on variables - where insertion occurs in a chromosome, what kinds of genes are relocated, and the type of cell involved • In bacteria, transposons are known to be involved in: - changes in traits i.e. colony morphology, pigmentation, and antigenic characteristics - replacement of damaged DNA - the transfer of drug resistance in bacteria Result of Transposon Activity in Bacteria: ** • changes in traits such as colony morphology, pigmentation, and antigenic characteristics • replacement of damaged DNA • transfer of drug resistance in bacteria

Mutations Terminology

Spontaneous Mutation: random change in the DNA arising from errors in replication that occur without a known cause Induced Mutation: result from exposure to known mutagens, which are primarily physical or chemical agents that damage DNA Substitution Mutation: changing of single base in the DNA code that may result in the placement of a different amino acid Frameshift Mutation: addition or deletion of bases that changes the reading of mRNA codons • A mutation that changes a single nucleotide can result in a different amino acid being added into a protein. • DNA mutations are passed on to a cell's progeny. • A mutation that causes a change in a single nucleotide in DNA changes the corresponding nucleotide in mRNA, resulting in a different codon.

Which of the following are directly needed in order for translation to occur?

mRNA tRNA rRNA Amino Acids

Components of an operon in a sequence of DNA

operator structural genes

Short lengths of RNA called __________ have the ability to control the expression of certain genes.

small interfering RNA (siRNA)

Regulation of Protein Synthesis and Metabolism: OPERON

• A major form of gene regulation in prokaryotes is through systems called Operons* • Genes are regulated to be active only when their products are required • In prokaryotes this regulation is coordinated by operons, a set of genes, all of which are regulated as a single unit (single operating site) • Operons - section of DNA that contains 1 or more structural genes along with a corresponding operator gene that controls transcription - permits genes for a particular metabolic pathway to be induced or repressed in unison by the same regulatory element - Either inducible or repressible - Category of operon is determined by how it is affected by the environment within the cell • Inducible Operons* - Many catabolic operons are Inducible* = the operon is turned on (induced) by the substrate of the enzyme for which the structural genes code - Enzymes are needed to metabolize a nutrient (i.e. Lactose) are produced only when that nutrient is present • Repressible Operons* - contain genes coding for anabolic enzymes such as those used to synthesize amino acids - Several genes in series are turned off (repressed) by the product synthesized by the enzyme

RNA Viruses with Reverse Transcriptase: Retroviruses

• A most unusual class of viruses can reverse the order of the flow of genetic information (Usual genetic patterns: DNA>DNA, DNA>RNA, RNA>RNA) • Retroviruses including HIV(cause of AIDS and HTLV 1) cause 1 type of human leukemia, synthesize DNA using their RNA genome as a template • They accomplish this by means of Reverse Transcriptase* that comes packaged with each virus particle • This enzyme synthesizes a single-stranded DNA against the viral RNA template and then directs the formation of a complementary strand of this ssDNA, resulting in a double-strand of viral DNA • dsDNA strand enters the nucleus, where it can be transcribed by the usual mechanisms into a new viral ssRNA and used to assemble new viral particles • When the DNA of some retroviruses becomes inserted into the host's DNA as a provirus, cells may be transformed and produce tumors • Insertion allows HIV to remain latent in an infected cell for several years

Mutations: Changes in the Genetic Code

• A permanent inheritable alteration in the DNA sequence of a cell is a mutation* - phenotype change that is due to an alteration in the genotype - On a strictly molecular level, a mutation is an alteration in the nitrogen base sequence of DNA • Wild Type /Wild Strain - A microorganism that exhibits a natural, nonmutated characteristic • Mutant Strain - if a microorganism develops a mutation - Can show variance in morphology, nutritional characteristics, genetic mechanisms, resistance to chemicals, temperature preference, and nearly any type of enzymatic function - Useful for tracking genetic events, unraveling genetic information, and pinpointing genetic markers - Simplest way to detect mutant bacteria - inoculate solid media containing differential or selective agents i.e. metabolic substrates or ATBs Isolating Mutants Replica plating technique allows identification of mutants: (a) Culture is exposed to mutagen (b) Isolated colonies are transferred on a master plate (c) tiny clump of cells are picked up and transferred to a plate with complete medium and another with incomplete medium (d) Colonies in the complete medium plate missing from the incomplete one are mutant colonies that can be sub-cultured for further use

Riboswitches

• ncRNAs in bacteria - shorter leader segments of mRNA that are trascribed but not expressed in a finished protein - Acting as a switch means that they can be turned on or off, depending on their folding pattern = they can both start or stop a step in gene expression - Unique ability to sense and bind a specific molecule (ligand) and translation can proceed - Regulates synthesis of some vitamins

Events in DNA Replication

• All chromosomes have a specific origin of replication site as the place where replication will be initiated • The origin of replication is AT-rich, thus less energy is required to separate the two strands • There are two replication forks where new DNA is being synthesized, each containing its own set of replication enzymes 1. The chromosome to be replicated is unwounded by a helicase, forming a replication fork with two template strands. 2. The template for the leading strand (blue) is oriented 3' to 5'. This allows the DNA polymerase III to add nucleotides in the 5' to 3' direction toward the replication fork, so it can be synthesized as a continuous strand. Note that direction of synthesis refers to the order of the new strand (red). 3. The template for the lagging strand runs 5' to 3' (opposite to the leading strand), so to make the new strand in the 5' to 3' orientation, synthesis must proceed backward, away from the replication fork. 4. Before synthesis of the lagging strand can start, a primase adds an RNA primer to direct the DNA polymerase III. Synthesis produces unlinked segments of RNA primer and new DNA called Okazaki fragments. 5. DNA polymerase I removes the RNA primers and fills in the correct complementary DNA nucleotides at the open sites. 6. Unjoined ends of the nucleotides (a nick) must be connected by a ligase.

Translation: The Second Stage of Gene Expression

• All of the elements needed to synthesize a protein, from the mRNA to the tRNAs with amino acids, are brought together on the ribosomes • This process occurs in 4 stages: - Initiation - Elongation - Termination - Protein folding and processing

Genotypes and Phenotypes

• All types of genes constitute the genetic makeup - genotype* • The expression of the genotype creates observable traits - phenotype* Ex) A person inherits a combination of genes (genotype) that gives a certain eye color or height (phenotype) - A bacterium inherits genes that direct the formation of a flagellum or the ability to metabolize a certain substrate - A virus has genes that dictate its capsid structure • A chromosome is subdivided into genes, the fundamental unit of heredity responsible for a given trait - Site on the chromosome that provides information for a certain cell function - Segment of DNA that contains the necessary code to make a protein or RNA molecule • Three basic categories of genes: - Genes that code for proteins - structural genes - Genes that code for RNA - Genes that control gene expression - regulatory genes

Additional Methods of Gene Expression

• Analogous gene control mechanisms in Eukaryotic cells are not as well understood but it is known that gene function can be altered by intrinsic regulatory segments similar to operons • Some molecules called Transcription Factors - insert on the grooves of the DNA molecule and enhance transcription of specific genes • Transcription factors can regulate gene expression in response to environmental stimuli i.e. nutrients, toxin levels, or even temp** • Regulated during growth and development**, leading to the 100s of different tissue types found in higher multicellular organisms • Regulatory RNA may exert control on several levels in both Prokaryotes and Eukaryotes*

Arginine Operon: Repressible

• Bacterial systems for synthesis of amino acids, purines and pyrimidines, and other processes work on a principle that is the reverse of the lac operon = Repression* • A metabolically active cell that is consuming large amounts of the amino acid arginine (arg) will serve to illustrate the operation of a repressible operon • The arg operon is set to on - arginine is being actively synthesized through the action of the operon's enzymatic products • In an active cell, the arginine will be used immediately and the repressor will remain inactive (unable to bind the operator) because there is too little free arginine to activate it • Cell's metabolism begins to slow down - the synthesized arginine is no longer used up and accumulates • Free arginine is then available to act as a corepressor by attaching to the repressor COREPRESSOR* - binds to an inactive repressor to make an active repressor. • This reaction changes the shape of the repressor, making it capable of binding to the operator and stopping transcription • Arginine will cease to be synthesized until the cell once again requires it in metabolism • Normally on and will be turned off when the product of the pathway is no longer required 1) Operon On: Arginine being used by cell A repressible operon remains on when its nutrient products (here, arginine) are in great demand by the cell. The repressor has the wrong shape to bind to the DNA operator without a corepressor, so that RNA polymerase is free to actively transcribe the genes and translation actively proceeds. • When excess arginine is present, it binds to the repressor and changes it. Then the repressor binds to the operator and blocks arginine synthesis. Arginine is the corepressor 2) Operon Off: Arginine building up The operon is repressed when (1) arginine builds up and, serving as a corepressor, activates the repressor. (2) The activated repressor complex affixes to the operator and blocks the RNA polymerase and further transcription of genes for arginine synthesis. • In a repressible operon, excess product acts as a corepressor to decrease* the transcription of the operon.**

Transfer RNA - tRNA

• Contains the anticodon and an amino acid binding site • Acts as a translator of the mRNA code into protein • 75 - 95 nucleotides in length bent into hairpin loops to form a cloverleaf structure further packed into a complex helix • Bottom loop of the cloverleaf exposes the tRNA specific anticodon complementary to a mRNA codon • Binding site for amino acids is specific for each anticodon b) Transfer RNA (tRNA) - Left: The tRNA strand loops back on itself to form intrachain hydrogen bonds. The result is a cloverleaf structure, shown here in simplified form. At its bottom is an anticodon* that specifies the attachment of a particular amino acid at the 3' end. Right: A three-dimensional view of tRNA structure.

Lactose (lac) Operon: Inducible Operon

• Control system that manages the regulation of lactose metabolism; composed of 3 DNA segments: 1. Regulator - gene that codes for repressor* (protein capable of repressing the operon); The protein product of a repressor gene is the repressor* which binds the operator to stop transcription. 2. Control locus - composed of promoter (recognized by RNA polymerase) and operator (sequence that acts as an on/off switch for transcription) 3. Structural locus - made of 3 genes each coding for an enzyme needed to catabolize lactose: - B-galactosidase - hydrolyzes lactose - Permease - brings lactose across cell membrane - B-galactosidase transacetylase - uncertain function • Two components of an operon include the operator* that acts as an on/off switch, and the structural* gene sequences.

Repair of Mutations

• DNA that has been damaged by UV radiation can be restored by Photoactivation or Light repair* • This repair mechanism requires visible light and a light sensitive enzyme, DNA photolyase, which can attach to sites of abnormal pyrimidine bonding and restore the original DNA structure • UV repair mechanisms are successful only for a relatively small # of UV mutations • Excision Repair* - Mutations can be excised by a series of enzymes that remove the incorrect bases and add the correct ones; Proteins that remove incorrect bases and replace them with correct ones. - 1st enzymes break the bonds between the bases and the sugar-phosphate strand at the site of the error - A different enzyme subsequently removes the defective bases one at a time leaving a gap that will be filled in by DNA polymerase I and Ligase - Repair system can also locate mismatched bases that were missed during proofreading Ex) C mistakenly paired with A, or G with T - The base must be replaced soon after the mismatch is made or it will not be recognized by the repair enzymes

Transcription: The First Stage of Gene Expression

• During transcription, an RNA molecule is synthesized using the codes on DNA as a guide or template • It proceeds in 3 stages: 1. Initiation: RNA polymerase binds to promoter region upstream of the gene - Promoter region consists of 2 sets of DNA sequences located just before the initiation site - Primary function of the promoter is to provide position for initial binding of the RNA polymerase - Sigma Factor - special protein molecule, guides the RNA polymerase to the correct position on the promoter - Prior to the first step of transcription, the RNA polymerase begins to seperate the 2 strands of the DNA helix and forms an open "bubble" for transcription (where nucleotides of mRNA will be assembled) - Only 1 strand of DNA - Template Strand* - is transcribed - will have reading frame oriented in the 3'-5' direction recognized by the RNA polymerase > message for the correct sequence of amino acids that will be linked during translation (protein synthesis) - Other strand - Nontemplate Strand* - sometimes called the coding strand* because its sequence is the same order as mRNA (but will have thymine instead of uracil) - not transcribed - Important triplet appearing early in DNA template is TAC* - will be transcribed into AUG on the mRNA which is the start Codon* (signals the location on the mRNA where translation starts) - Promoter sequences are not transcribed as part of the final mRNA molecule 2. Elongation: RNA polymerase adds nucleotides complementary to the DNA template strand in the 5′ to 3′ direction (Uracil (U) is placed complementary to adenine (A) 3. Termination: RNA polymerase recognizes a "STOP" sign in the DNA and releases the transcript (100-1,200 bases long) Functions of RNA polymerase: - Synthesizes an RNA molecule from DNA template - Unwinds the DNA so that transcription can take place

Gene-Protein Connection

• Each structural gene is an ordered sequence of nucleotides that codes for a protein's primary structure • Groups of three consecutive bases, triplets or codons, on one DNA strand are transcribed into RNA sequence triplets • Each triplet of nucleotides on the RNA specifies a particular amino acid • A protein's primary structure (chain of amino acids) determines its shape and function • Proteins contribute to the cell phenotype as enzymes and structural proteins

Replication, Transcription, and Translation of dsDNA Viruses

• Early phase - viral DNA enters the nucleus, where several genes are transcribed into a messenger RNA • Next, the mRNA transcript then moves into the cytoplasm to be translated into viral proteins (enzymes) needed to replicate the viral DNA - usually occurs in the nucleus with the host cell's own DNA polymerase (some viruses have their own i.e. herpes) • Late phase - other viral genes are transcribed into proteins required to form the capsid and other structures • The new viral genomes and capsids are assembled, and the mature viruses are released by budding or cell disintegration • Double-stranded DNA viruses can interact directly with the DNA of their host cell • Some viruses, the viral DNA becomes silently integrated into the host's genome by insertion at a particular site on the host genome • Persistence of viral DNA may also lead to transformation of the host cell into a cancer cell • Several DNA viruses (i.e. Hep B/HBV, herpesviruses, papillomaviruses) are known to be initiators of cancers and are termed Oncogenic** • The mechanisms of oncogenic transformation involve viral genes that regulate cellular genomes and control the cell division** • The virus penetrates the host cell and releases DNA: 1) DNA enters the nucleus 2) DNA is transcribed by the host cell enzymes into mRNA 3) Viral mRNA leaves the nucleus and is translated into structural proteins - proteins are transported into the nucleus 4) Viral DNA is replicated repeatedly in the nucleus 5) Viral DNA and proteins are assembled into a mature virus in the nucleus 6) Because it is double-stranded, some viral DNA can insert itself into host DNA (latency); some exceptions occur i.e. poxviruses

Ribosomal RNA - rRNA

• Forms a complex cellular structure that contributes to the process of translation • The prokaryotic (70S) ribosome is a particle composed of tightly packaged ribosomal RNA (rRNA) and protein • Forms complex three-dimensional figures that contribute to the structure and function of ribosomes: reading the mRNA code, facilitating its interaction with tRNA, and producing proteins at an impressive rate • A metabolically active bacterial cell can accommodate up to 20,000 of the tiny factories

Applications of the DNA Code

• Genetic information flows from DNA to RNA to protein • Master code of DNA is first used to synthesize RNA via a process called Transcription* (DNA>RNA) - the information contained in the RNA is then used to produce proteins in a process known as Translation* - The flow of genetic information in a cell is a process starting with DNA which encodes RNA which encodes protein • Principal exceptions to this pattern are found in RNA viruses which convert RNA to other RNA; and in retroviruses which can convert RNA to DNA • A wide variety of specialized RNAs act by regulating gene function

Positive and Negative Effects of Mutations

• Many mutations are not repaired, cell copes depending on the nature of the mutation and the strategies available to that organism • Mutations are passed on to the offspring of organisms during reproduction and to new viruses during replication - they become a long-term part of the gene pool • Most mutations are harmful to organisms but some can provide adaptive advantages • If mutation leading to nonfunctional protein occurs in a gene for which there is only a single copy (i.e. Haploid organisms) the cell wall will probably die • Mutations leading to nonfunctional proteins are harmful, possibly fatal • Organisms with mutations that are beneficial in their environment can readily adapt, survive, and reproduce - these mutations are the basis of change in populations • Any change that confers an advantage during selection pressure will be retained by the population

DNA Recombination Events

• Refers to the transfer of genes from donor to recipient microorganisms, where the recipient strain shows a change in genetic makeup at the end.* • Recombination - an event where 1 bacterium donates DNA to another bacterium is a type of genetic transfer, the end result is a new strain different from both the donor and the original recipient strain • Recombinant - any organism that has acquired genes that originated in another organism • DNA transfer between bacterial cells involve pieces of DNA in the form of Plasmids*/chromosomal fragments • When one bacterium donates DNA to another bacterium, a type of genetic recombination known as horizontal gene transfer** has occurred. • Plasmids* - small circular pieces of DNA that contain their own origin of replication and can replicate independently; found in many bacteria, some fungi, and contain a few dozen genes; often carry adaptive genes i.e. for drug resistance - found in the cytoplasm of prokaryotes that is capable of independent replication and usually contains nonessential genes • 3 means for genetic recombination in bacteria: - Conjugation - Transformation - Transduction

Categories of Mutations

• Mutations range from large mutations (long genetic sequences are gained or lost) to small (affect only a few bases on a gene) • Point mutation - addition, deletion, or substitution of a few bases • Missense Mutation - any change in the code that leads to placement of a different amino acid - Create a faulty, nonfunctional (or less functional) protein - Produce a protein that functions in a different manner - Cause no significant alteration in protein function • Nonsense mutation - changes a normal codon into a stop codon that does not code for an amino acid and stops the production of the protein wherever it occurs - Almost always results in a nonfunctional protein • Silent mutation - alters a base but does not change the amino acid - Ex) Because of redundancy of the code, ACU, ACC, ACG, and ACA all code for threonine so a mutation that changes only the last base will not alter the sense of the message in any way • Back-mutation - occurs when a gene that has undergone mutation reverses (mutates back) to its original base composition • Inversion - occurs when 2 nucleotides have switched their order, which nearly always change the amino acid by inverting GUC (valine) to GCU (threonine) • Frameshift mutation - when the reading frame of the mRNA is altered, shifts to the left or right; when 1 or more bases are inserted into or deleted from a newly synthesized DNA strand - Nearly always result in a nonfunctional protein because the codon structure will become reset from that point on, will code for a different sequence of amino acids from the original DNA and is likely to introduce stop codons - Insertion or deletion of bases in multiples of 3 (3,6,9etc) does not disturb the reading frame downstream from the mutation site - It can still disrupt the structure of the protein depending on the change in amino acid sequence

Lac Operon

• Normally off - In the absence of lactose, the repressor binds with the operator locus and blocks transcription of downstream structural genes 1) Operon Off: No Lactose In the absence of lactose, a repressor protein (encoded by the regulatory gene) - relatively large molecule is allosteric (its activity can be altered depending which active site and substrate are in play); - If an operon's repressor is in its active form that means Transcription from the operon is not occurring - A substrate binding to one site can distort a different site and prevent it from accepting its substrate - In absence of Lactose, the repressor protein interacts with the operator and causes the operator to distort into a temporary loop configuration - Loop blocks access of the RNA polymerase to the DNA of the operator and prevents transcription Ex) Repressor is a "lock" on the operator, if the operator is locked, the structural genes cannot be transcribed - The regulator gene is located in a separate site from the operator region and is not affected by this block on the operon - Suppression of transcription (and translation) prevents the unnecessary synthesis of enzymes for processing lactose. • Lactose turns the operon on by acting as the inducer -Binding of lactose to the repressor protein changes its shape and causes it to fall off the operator. - RNA polymerase can bind to the promoter and begin transcription - Structural genes are transcribed in a single unbroken transcript coding for all 3 enzymes - During translation, each protein is synthesized separately - Lactose is ultimately responsible for stimulation the chain of events leading to protein synthesis = Inducer* 2) Operon on: Lactose Present - Upon entering the cell, the substrate (lactose) becomes a genetic inducer by attaching to the repressor, which is render inactive and falls away. The operator is no longer closed off and its DNA becomes accessible to the RNA polymerase. The RNA polymerase transcribes the structural genes, and the mRNA is translated into enzymes that can act on the lactose substrate. • Lac operon - functions only in the absence of glucose or if the cell's energy needs are not being met by available glucose - Glucose is the preferred carbon source* because it ca be used immediately in growth and does not require induction of an operon - When glucose supply is present, a 2nd regulatory system ensures that the lac operon is inactive, regardless of lactose levels in the environment

Initiation of Translation

• Prokaryotic Cells - the mRNA molecule leaves the DNA transcription site and is transported directly to ribosomes • Ribosomal subunits are assembled in a way that forms sites to hold the mRNA and tRNAs • Ribosome recognizes these molecules and stabilizes reactions between them • Small subunit holds the tRNAs and is actively involved in peptide bond formation by means of specialized ribozyme (RNA based catalyst) • Small subunit of the ribosome binds at a specific site on the mRNA and places the start codon (AUG) in correct alignment with the P site • With mRNA message in place on the assembled ribosome, the next step in translation involves entrance of tRNAs with their amino acids • Pool of cytoplasm around the region contains a complete array of tRNAs, previously charged by having the correct amino acid attached • Complementary tRNA meets with mRNA code is guided by the 2 sites on the large subunit of the ribosome called the P site (left) and the A site (right) • These sites are like recessed spaces tucked within the 2 subunits of the ribosome with each site accommodating a tRNA • The ribosome also has an exit or E site where used tRNAs are released

DNA Replication Terminology

• RNA Primers - Short segment of RNA that serves as binding sites for DNA polymerase to start adding nucleotides to the new strand of DNA • Replication Forks - Y-shaped point on a replicating DNA molecule where the DNA polymerase is synthesizing new strands of DNA • Leading Strand - The newly forming DNA strand that is replicated in a continuous fashion w/o fragments because it is oriented in the correct 3'-5' direction for the DNA polymerase • Lagging Strand - The newly forming DNA strand that is discontinuously replicated in short segments (Okazaki fragments) because the template cannot be continuously read by the DNA polymerase • Okazaki Fragments - In replication of DNA, a segment formed on the lagging strand where biosynthesis is conducted in a discontinuous manner as required by the DNA polymerase orientation • DNA Polymerase I - Enzyme responsible for the replication of DNA, several versions of the enzyme exist, each completing a unique portion of the replication process • Ligase - An enzyme required to join nucleotides together to complete the final attachment of the ends of 2 fragments of DNA

Major Types of Ribonucleic Acid Involved In Protein Synthesis

• RNA Type - Contains Codes For - Function in Cell - Translated: • Messenger (mRNA) - Sequence of amino acids in protein - Carries the DNA master code to the ribosomes - Yes • Transfer (tRNA) - Specifying a given amino acid - Carries amino acids to ribosomes during translation - No • Ribosomal (rRNA) - Several large structural rRNA molecules - Forms the major part of ribosomes and participates in protein synthesis - No • Primer - An RNA that can begin DNA replication - Primes DNA - No

Replication, Transcription, and Translation of RNA Viruses

• RNA viruses exhibit several differences from DNA viruses; genomes enter the host cell already in an RNA form and the virus cycle occurs entirely in the cytoplasm for most viruses • RNA viruses can have on of the following genetic messages: 1. a positive-strand (+) genome that comes ready to be translated into proteins 2. a negative-strand (-) genome that must be converted to a positive-strand before translation 3. a positive-strand (+) genome that can be converted to DNA or a dsRNA genome

The Master Genetic Code

• Represented by mRNA codons and their specific amino acids • Code is universal among organisms and redundant • Once the mRNA codon is known, the original DNA sequence, the complementary tRNA code, and the types of amino acids in the protein are automatically known • Cannot predict (backward) from protein structure what the exact mRNA codons are because of a factor called Redundancy* or Degeneracy* = a particular amino acid can be coded for by more than 1 codon•

Conjugation

• Requires the attachment of 2 cells and the formation of a bridge that can transport DNA • Transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via direct contact* • Gram (+) and Gram (-) can conjugate but only Gram (-) cells operate with a specialized plasmid called Fertility (F Factor)* - directs the synthesis of a unique Pilus (sex pilus) that functions in most conjugation transfers • Donor (F+ cell) transfers fertility plasmid through pilus to recipient (F- cell), which becomes F+ cell • Some F+ cells become Hfr cells (high frequency of recombination) • F factor - the specialized plasmid that directs conjugation in gram-negative cells* • Conjugation in bacteria refers to horizontal gene transmission via pili* (1) The pilus of donor cell (top) attaches to receptor on recipient cell and retracts to draw the two cells together. This is the mechanism for gram-negative bacteria. • High-frequency recombination - donor's fertility plasmid is integrated into the bacterial chromosome • When conjugation occurs, a portion of the chromosome and a portion of the fertility plasmid are transferred to the recipient (2) Transfer of F factor, or conjugative plasmid (3) High frequency (Hfr) transfer involves transmission of chromosomal genes from a donor cell to a recipient cell. The donor chromosome is duplicated and transmitted in part to a recipient cell, where it is integrated into the chromosome. • Resistance (R) Plasmids / Factors - bear genes for resisting antibiotics and other drugs are commonly shared among bacteria through conjugation

Translation

• Ribosomes assemble on the 5′ end of an mRNA transcript • Ribosome scans the mRNA until it reaches the start codon, usually AUG (formyl methionine - Met) • A tRNA molecule with the complementary anticodon and methionine amino acid enters the P site of the ribosome and binds to the mRNA (1) Entrance of tRNAs 1 and 2 • A second tRNA with the complementary anticodon fills the A site • A peptide bond is formed between the amino acids on the neighboring tRNAs (2) Formation of peptide bond • The first tRNA is released and the ribosome slides down to the next codon (3) Discharge of tRNA 1 at E site • Another tRNA fills the A site and a peptide bond is formed (4) First translocation; tRNA 2 shifts into P site; tRNA 3 enters ribosome at A (5) Formation of peptide bond • This process continues until a stop codon is reached (6) Discharge of tRNA 2; second translocation; tRNA 4 enters ribosome (7) Formation of peptide bond

Translation Termination

• Termination of protein synthesis is brought about by the presence of at least 1 special codon occurring just after the codon for the last amino acid • Termination codons - UAA, UAG, UGA - are codons for which there is no corresponding tRNA • Termed Stop Codons* - carry a necessary message = STOP HERE - when reached, a special enzyme breaks the bond between the final tRNA and the finished polypeptide chain, releasing it from the ribosome • Before the peptide chain is released from the ribosome, it begins folding upon itself to achieve its biologically active conformation • Other alterations - Post-translational Modifications* • Some proteins must have the starting amino acid (formyl methionine) clipped off • Proteins destined to become complex enzymes have cofactors added and some join with other completed proteins to form Quaternary Levels* of structure • Protein synthnesis in bacteria is efficient and rapid • At 37C, 12-17 amino acids per second are added to growing peptide chain • An average protein consisting of about 400 amino acids requires less than half a minute for complete synthesis • Further efficiency is gained when the translation of mRNA starts while transcription is still occurring • A single mRNA is long enough to be fed through more than 1 ribosome simultaneously • This permits the synthesis of 100s of protein molecules from the same mRNA transcript arrayed along a chain of ribosomes • Polyribosomal Complex* - assembly line for mass production of proteins • Protein synthesis consumes an enormous amount of energy - approx. 1200 ATPs or ATP equivalent are consumed for synthesis of an average-size protein

Packaging of DNA

• The DNA molecule is compacted in the cell by supercoils, or superhelices • Prokaryotes (simpler system) - circular chromosomes is packaged by the action of a special enzyme called Topoison erase (DNA Gyrase*) - DNA Gyrase - coils the chromosome into a tight bundle by introducing a reversible series of twists into the DNA molecule • Eukaryotes (complex system) - 3 or more levels of coiling - DNA molecule of a chromosome is linear, is wound twice around the histone proteins = chain of Nucleosomes* - Nucleosomes fold in a spiral formation upon one another - Greater supercoiling occurs when spiral arrangement further twists on its radius into giant spiral loops radiating from the outside (makes chromosome visible during mitosis)

Transformation

• The acceptance by a bacterial cell of small DNA fragments from the surrounding environment* Chromosome fragments from a lysed cell are accepted by a recipient cell; the genetic code of the DNA fragment is acquired by the recipient • Donor and recipient cells can be unrelated • Useful tool in recombinant DNA technology • DNA fragment (blue) delivering cap+gene for capsule formation (red) binds to a surface receptor on a competent recipient cell. • DNA is converted to one strand and transported into the cell, by the DNA transport system. • The DNA strand is incorporated into the recipient chromosome. • Recipient is now transformed with gene for synthesizing a capsule.

DNA Replication

• The sequence of bases along the length of DNA constitutes its "language" • For this language to be preserved for 100s of generations, the codes it contains will need to be duplicated with high fidelity = process of duplication is called DNA Replication* • Replication occurs on both strands simultaneously • Must occur prior to cell division to ensure that each new cell has a complete set of DNA chromosomes • Semiconservative Replication**: 1. The parent DNA molecule is uncoiled 2. The two strands are separated exposing the nucleotide sequence to serve as templates 3. Two new complementary strands are synthesized by using each single-stranded template as pattern - The DNA is duplicated prior to binary fission. • Replication can be very rapid - must be completed in a single generation time (around 20 min for E.Coli) • DNA replication requires careful orchestration of the actions of 30 different enzymes that separate the strands of existing DNA, copy its template, and produce 2 complete daughter molecules

Genetics

• The study of heredity/inheritance of biological characteristics by life forms • The science of genetics explores: - Transmission of biological traits from parent to offspring - Expression and variation of those traits - Structure and function of genetic material - How this material changes

Eukaryotic Transcription and Translation

• They are not colinear • Introns* - located within their genes, do not code for protein; are interspersed between coding regions called Exons* (will be translated into protein 1. Do not occur simultaneously - transcription occurs in the nucleus and translation occurs in the cytoplasm 2. Eukaryotic start codon is AUG, but it does not use formyl-methionine 3. Eukaryotic mRNA encodes a single protein, unlike bacterial mRNA which encodes many 4. Eukaryotic DNA contains introns- intervening sequences of noncoding DNA - which have to be spliced out of the final mRNA transcript Illustration (words to remember as ex): • Prokaryotic gene - SAM SAW HIS NEW CAR GET HIT • Eukaryotic gene that codes for the same portion would read: SAM SAW SVXF FPL HIS NEW CAR QZWVP GET HIT • The recognizable words are the exons, while the other letters represent the introns • Discontinuous genetic structure (Split Gene) - requires further processing before translation • Transcription of the entire gene with both exons and introns occurs 1st = producing a pre-mRNA • Next RNA-protein complex called a Spliceosome* recognizes the exon-intron junctions and enzymatically cuts through them in a process called RNA splicing • Action of splicer enzyme loops the introns into lariat-shaped pieces, excises them, and joins the exons end to end = a strand of mRNA with no intron material is produced > completed mRNA strand can then proceed to the cytoplasm to be translated Different types of introns that do not code for cell substances: Ex) Certain introns have been found to code for an enzyme called reverse transcriptase which can convert RNA into RNA - Other introns are translated into endonucleases, enzymes that can snip DNA and allow insertions and deletions into DNA sequence - Some introns have innate catalytic function and can splice themselves out of an RNA transcript - Non-protein-coding DNA is vital for cell function • Human DNA has an average of 8 introns per gene = represents a significant % of DNA found in chromosomes

The Structure of DNA: Double Helix

• To analyze the structure of DNA, magnify gene about 5 mil. x - James Watson, Francis Crick (1953) - determined that DNA is a giant molecule w/ 2 strands bound together into double helix; universal structure except in some viruses (that contain single-stranded DNA). • Basic unit of DNA structure is a nucleotide, composed of phosphate, deoxyribose sugar, and a nitrogen base: - A deoxyribose sugar - A phosphate group - A nitrogenous base: adenine (A), guanine (G), thymine (T), cytosine (C) • Nucleotides covalently bond to form a sugar-phosphate backbone • Nitrogenous bases (Purines and Pyrimidines*) covalently bond to the 1′ carbon of each sugar and span the center of the molecule to pair with a complementary base on the other strand = forms double helix • Paired bases are held together by hydrogen bonds that can be disrupted/"unzipped" into 2 strands which allows access to info encoded in the nitrogen base sequence - The purine Adenine (A) pairs with the pyrimidine Thymine (T) - The purine Guanine (G) pairs with the pyrimidine Cytosine (C) - Adenine (A) to thymine (T) with 2 hydrogen bonds - Guanine (G) to cytosine (C) with 3 hydrogen bonds • An average bacterial chromosome consists of 5-6 mil nucleotides; the 46 chromosomes (diploid) of humans total about 6.4 billion nucleotides • Double Helix - 1 side runs in the opposite direction of the other = Antiparallel Arrangement* - 1 helix runs from the 5'-3' direction and the other runs from the 3'-5' direction - significant factor in DNA synthesis, transcription, and translation - Each strand provides a template for the exact copying of a new strand - Order of bases constitutes the DNA code

The Ames Test

• To determine the carcinogenic potential of a chemical • Experimental subjects are bacteria whose gene expression and mutation rate can be readily observed and monitored • More rapid than animal testing • Any chemical capable of mutating bacterial DNA can similarly mutate mammalian DNA • Agricultural, industrial, and medicinal compounds are screened using the Ames test • Indicator organism is a mutant strain of Salmonella typhimurium that has lost the ability to synthesize histidine • This mutation is highly susceptible to back-mutation

Messenger RNA - mRNA

• Transcribed version of a structural gene or genes in DNA • Synthesized following complementary-base pairing by a process similar to synthesis of the leading strand during DNA replication • Message is in triplets called codons a) Messenger RNA (mRNA) - A short piece of messenger RNA (mRNA) illustrates the general structure of RNA: single strandedness, repeating phosphate-ribose sugar backbone attached to single nitrogen bases; use of uracil instead of thymine

Interpreting the DNA Code

• Transcription produces mRNA complementary to the DNA gene • During translation, tRNAs use their anticodon to interpret the mRNA codons and bring in the specific amino acids • If the DNA sequence is known, the mRNA codon can be surmised • If a codon is know, the anticodon and amino acid sequence can be determined • Reverse is not possible due to redundancy code • mRNA codons and their corresponding amino acids specificities are given • There are 64 different triplet codes2 and only 20 different amino acids = some amino acids are represented by several codons Ex) Leucine and Serine can each be represented by any 6 different triplets but Tryptophan and Methionine are represented by a single colon • Codons as leucine, only the 1st 2 nucleotides are required to encode the correct amino acid, and the 3rd nucleotide does not change its sense = Wobble* - thought to permit some level of variation or mutation w/o changing the message

Major Participants in Transcription and Translation

• Transcription, the formation of RNA using DNA as a template • Translation, the synthesis of proteins using RNA as a template, are highly complex • A number of components participate: most prominently messenger RNA, transfer RNA, ribosomes, several types of enzymes, and a storehouse of raw materials • The main enzyme responsible for transcription is RNA polymerase.

Genetics of Animal Viruses

• Viruses essentially consist of 1 or more pieces of DNA or RNA enclosed in a protective coating * • Genetic parasites that require access to their host cell's genetic and metabolic machinery to be replicated, transcribed, and translated, also have potential for genetically changing the cells • Because they contain mainly those genes needed for production of new viruses, the genomes tend to be very small and compact • Viruses show extensive variety in genetic patterns - the nucleic acid is linear in form; in others, it is circular • Genome of most viruses exist in a single molecule, though in a few, it is segmented into several smaller molecules • Viral genome - one or more pieces of DNA or RNA that contain only genes needed for production of new viruses • Viruses require access to host cell's genetics and metabolic machinery to instruct the host cell to synthesize new viral particles • Most viruses contain normal double-stranded (ds) DNA or single-stranded (ss) RNA, but other patterns exist** • In all viruses, viral mRNA is translated into viral proteins on host cell ribosomes using host tRNA** • With few exceptions, replication of the viral DNA occurs in the nucleus. The genome of most RNA viruses is replicated in the cytoplasm

Size Genomes

• Viruses have from a few to over 1000s genes Ex) E. Coli - has a single chromosome containing 4288 genes; 1 mm; 1,000X longer than the cell A Human Cell - is 5x that into 46 chromosomes; - 46 chromosomes containing 31,000 genes; 6 feet; 180,000X longer than the cell • Complex coiling of the DNA chain* allows such elongated genomes fit into the miniscule volume of a cell (eukaryotes even smaller in the nucleus)

Positive-Strand Single-Stranded RNA Viruses

• ssRNA of positive-strand viruses i.e. polio come ready to be translated - it is immediately translated into a large protein and cleaved into individual functional units • One of these RNA polymerase that initiates the replication of the viral strand • Replication of a positive-message strand happens in 2 steps: 1. A negative strand is synthesized using the positive strand as a template by the usual base-pairing mechanisms 2. The resultant negative strand becomes a master template that formats the formation of new positive daughter strands 3. Further translation of the viral genome produces large numbers of structural proteins for final assembly and maturation of the viruses