Micro Test 2: Chapter 7 (Microbial Genetics)
Translation
-Code: sequence of bases in DNA -Codon: 3 bases in mRNA specify for one amino acid -3 bases in DNA: codon in mRNA (3 bases)- one amino acid in polypeptide -Genetic Code: 64 codons Start Codon: AUG Stop Codon: UAA, UAG, UGA Reading Frame: read code in groups of 3 bases during translation Translation process: -in 5' to 3' direction -Ribosomes coordinate and bring together mRNA and tRNA -Initiation: mRNA binds to small subunit; first tRNA-met base pair with start codon AUG -Large subunit binds -tRNA fits into P site -A site ready to receive next tRNA -Elongation: next tRNA fits in A site, base pairs with mRNA codon -peptide bond between amino acids -translocation: ribosome moves, tRNA- polypeptide in P site -Free tRNA released -A site ready to receive next tRNA -Repeat for each amino acid -Termination: stop codon in A site -releasing factors involved -polypeptide is released -ribosome complex dissociates -mRNA translated again or destroyed LOOK IN BOOK
Continued
-In DNA replication, the new polymer is also DNA -in protein synthesis, the new polymer is a particular type of RNA called messenger RNA, which then serves as a second template that dictates the arrangement of amino acids in a protein -some proteins form the structure of a cell, others (enzymes) regulate its metabolism, and still others transport substances across a membrane -in the overall process of protein synthesis, the synthesis of mRNA from a DNA template is called transcription, and the synthesis of protein from information in mRNA is called translation -by analogy, transcription transfers information from one nucleic acid to another as you might transcribe handwritten sentences to typewritten sentences in the same language. -translation transfers information from the language of nucleic acids to the language of amino acids as you might translate sentences into another language. -there are even proof reading enzymes that try to eliminate any errors that occur, ensuring that a correct copy is passed on. -in the case of viruses that have RNA as their genetic material, scientists were initially unable to understand how these viruses could make more RNA -then, the discovery of enzymes for reverse transcription revealed a press whereby RNA can make DNA -this DNA can then make more RNA -such viruses are known as retroviruses because of this reverse process -HIV, is a retrovirus -reverse transcription is a less accurate process than regular transcription -uncorrected errors are passed on as mutations, or permanent changes in the genes of an organism -HIV has a mutation rate of 500 times higher than that of most organisms -DNA replication, transcription, and translation all transfer information from one molecule to another -These processes allow information in DNA to be transferred to each new generation of cells and to be used to control the functioning of cells through protein synthesis
DNA Structure
-Nucleotide made of ribose, phosphate and nitrogenous base -Nitrogenous bases: Adenine, Gunaine, Thymine, Cytosine -Double Helix: 2 antiparalell strands held by hydrogen bonds between the bases -Always A-T, G-C -Chains grow from 5' to 3' -nucleotide attaches at 3' -phosphate is attached at carbon #5 of ribose -carbon #3 is free for next nucleotide to attach by its phosphate -DNA can replicate itself by complementary base pairing Figure 7.1 -the two upright strands, composed of sugar deoxyribose, and phosphate groups, are held together by hydrogen bonding between complementary bases. -Adenine always pairs with thymine, and guanine always pairs with cytosine -each strand can thus provide the information needed for the formation of a new DNA molecule -the DNA molecule is twisted into a double helix -the two sugar phosphate strands run in opposite (antiparallel) directions -each new strand grows from the 5' end toward the 3' end
Phenotypic Variation
-Phenotypic variations produced by mutations can be alterations in colony morphology, nutritional requirements, or temperature sensitivity. -mutations that alter nutritional requirements generally increase the nutritional needs of an organism, usually by impairing the organism's ability to synthesize one or more enzymes ---as a result the organism may require certain amino acids or vitamins in its medium because it can no longer make them itself -Auxotrophs: nutritionally deficient mutants, they require special substances in their medium to maintain growth. -Prototrophs: normal nonmutant forms, wild types -Comparison characteristics of these show the effects of mutations on metabolism. -another type of phenotypic variation of genetic origin is temperature sensitivity -some phenotypic variations are caused by environmental factors and occur without any change in the genotype -for example, large amounts of sugar or irritants in the medium can cause some organisms to form a larger than normal capsule -some organisms such as anthrax bacterium, form spores in open air, in spilled blood, or on tissue surfaces but not inside tissues -variations in environmental temperature can affect pigment synthesis -serratia marcescens usually produces pigment at room temperature but may not do so at higher temperatures -it has the gene for pigment production, but the gene is expressed only at certain temperatures
Ribsomes (7.7)
-Site of protein synthesis -Two subunits of rRNA, proteins make functional ribosome -Ribosomal RNA (rRNA) binds closely to certain proteins to form two kinds of ribosome subunits -a subunit of each kind combines to form a ribosome -recall that ribosomes are sites of protein synthesis in a cell -they serve as binding sites for transfer RNA, and some of their proteins act as enzymes that control protein synthesis -prokaryotic ribosomes are made of a small (30S) and a large (50S) subunit -eukaryotic ribosomes are formed from a 40S and a 60S subunit -after the two subunits join together around the strand of mRNA, the synthesis of a peptide begins -the newly formed polypeptide chain grows out through a tunnel in the 50S subunit
Point Mutations
-a base substitution, or nucleotide replacement, in which one base is substituted for another at a specific location in a gene -the mutation changes a single codon in mRNA, and it may or may not change the amino acid sequence in a protein -suppose a three base sequence of DNA is changed from AAA to AAT -during transcription the mRNA codon will change from UUU to UUA -recall that uracil in RNA pairs with adenine in DNA -when the info in the mRNA is used to synthesize protein, the amino acid leucine will be substituted for phenylalanine in the protein -because of the single amino substitution, the new protein will be different from the normal protein -the effects on the phenotype of the organism will be negligible if the new protein functions as well as the original one -they will be significant if the new protein functions poorly or not at all -in rare instances the new protein may function better and produce a phenotype that is better adapted to its environment then the original phenotype -should the code in DNA be changed from AAA to AAG, the mRNA code becomes UUC instead of UUU -because the UUC and UUU codons both code for phenylalanine, the mutation has no effect on the protein being synthesized -in this case, it has long been thought that although genotype has changed, the phenotype is unaffected -however, we now know that such mutations are not "silent" -they do result in slightly different shifts in percentage of products -sometimes the substitution of a single base in DNA produces a terminator codon in mRNA -if the terminator codon is introduced in the middle of a molecule of mRNA destined to produce a single protein, synthesis will be terminated part way through the molecule -a polypeptide that will most likely be unable to function in the cell will be released, and the appropriate protein will not be synthesized -if the missing protein is essential to cell structure or function, the effect can be lethal
Mutations
-a change in sequence of nucleotides in DNA -Wild Type: organism isolated from nature -Mutant: organism with altered DNA -Auxotroph: nutritionally deficient mutant -Genotype: all genes -Phenotype: characteristics due to expression of genes -Mutations: important in evolutionary changes of microorganisms- development of strains
Frameshift mutation
-a mutation in which there is a deletion or insertion of one or more bases -such mutations alter all the three base sequences beyond the deletion or insertion -when mRNA transcribed from such altered DNA is used to synthesize a protein, many amino acids, in the sequence may be altered (remember, a ribosome reads an mRNA in codons, sets of three bases) -such mutations also commonly introduce terminator codons and cause protein synthesis to stop when only a short polypeptide has been made -frameshift mutations usually prevent synthesis of a particular protein, and they change both the phenotype and genotype -their effect on the organism depends on the role of the missing protein in the organism's function Deletion or insertion of one ore more bases in DNA: changes entire sequence of codons and greatly alters amino acid sequence; can introduce terminator codon and produce useless polypeptides instead of normal proteins. -Extensive missense -Immediate nonsense
Transcription
-all cells must constantly synthesize proteins to carry out their life processes; reproduction, growth, repair, and regulation of metabolism -this synthesis involves the accurate transfer of linear information of the DNA strands (genes) into a linear sequence of amino acids in proteins -to set the stage for protein synthesis, hydrogen bonds between bases in DNA strands are broken enzymatically in certain regions so that the strands separate -short sequences of unpaired DNA bases are thus exposed to serve as templates in transcription -only one strand directs the synthesis of mRNA for any one gene; the complementary strand is used as a template during DNA replication or during transcription of some other gene -recall that RNA contains the base uracil instead of thymine -thus, when mRNA is transcribed from DNA, uracil pairs with adenine; otherwise, the bases pair just as they do in DNA replication -messenger RNA is formed in the 5' to 3' direction -to transcribe its DNA, a cell must have sufficient quantities to nucleotides that contain high energy phosphate bonds, which provide energy for the nucleotides to participate in subsequent reactions -after separating the DNA strands, the enzyme RNA polymerase binds to one strand of exposed DNA recognizing a sequence of nucleotide bases in the DNA that indicates this is the start of a gene (promoter sequence) -after an enzyme binds to the first base in DNA (adenine, in this case), the appropriate nucleotide joins the DNA base enzyme complex -the new base then attaches by base pairing to the template base of DNA -the enzyme moves to the next DNA base, and the appropriate phosphorylated nucleotide joins the DNA base enzyme complex -the new base then attaches by base pairing to the template base of DNA -the enzyme moves to the next DNA base, and the appropriate phosphorylated nucleotide joins the complex -the phosphate of the second nucleotide is linked to the ribose of the first nucleotide , and pyrophosphate (two attached molecules of phosphate) is released -this forms the first link in a new polymer of RNA -energy to form this link comes from the hydrolysis of ATP and the release of two more phosphate groups -this process is repeated until the RNA molecule is completed -in prokaryotes, transcription and translation both take place in the cytoplasm, whereas in eukaryotes, transcription takes place in the cell nucleus -the mRNA of eukaryotic transcription must be completely formed and transported through the nuclear envelope to the cytoplasm before translation can begin -moreover, the mRNA molecule undergoes additional processing before it is ready to leave the nucleus -in eukaryotic cells, as well as in certain types of bacteria known as archaea, the regions of genes that code for proteins are called exons -exons are typically separated within a gene by DNA segments that do not code for proteins -such noncoding intervening regions are called introns -in the nucleus, RNA polymerase first forms mRNA from the entire gene, including all eons and introns -the newly formed, long mRNA molecule is streamlined by other enzymes, which remove the introns and splice together the eons -the resulting mRNA is ready to direct protein synthesis and to leave the nucleus
Nucleic Acids in Information Storage and Transfer: Information Storage
-all info for the structure and functioning of a cell is stored in DNA -for example in the chromosome of E.Coli, each of the paired strands of DNA contains about 5 million bases arranged in a particular linear sequence -the info in those bases is divided into units of several hundred bases each -each of these units is a gene -we might think of a gene as a sentence in the language of nucleic acids -each sentence in this language is constructed from a four lettered alphabet corresponding to the four nitrogenous bases in DNA (A,C,T,G) -when these four letters combine to make "sentences" several hundred letters along, the number of possible sentences becomes almost infinite -likewise, an almost infinite number of possible genes exists -if each gene contained 500 bases, a chromosome containing 5 million bases could contain 10000 different genes, thus information storage capacity of DNA is HUGE -haemophilus influenzae is the first microbe to have its genome completely sequenced, this was published in 1995
An Overview of Genetic Processes The Basis of Heredity
-all information necessary for life is stored in an organisms genetic material, DNA, or for many viruses, RNA. -Heredity: the transmission of this information from an organism to its progeny (offspring)--we must consider the nature of chromosomes and genes. -Chromosome: is typically a circular (in prokaryotes) or linear (in eukaryotes), threadlike molecule of DNA. -recall that DNA consists of a double chain of nucleotides with each nucleotide made up of a sugar, a phosphate, and a base (adenine, thymine, guanine, or cytosine) -the nucleotides are arranged in a helix, with the nucleotide base pairs held together by hydrogen -the specific sequence of nucleotides in the DNA can be copied to make another molecule of DNA or used to make RNA that then does protein synthesis.
Replication (teacher notes)
-at point of origin strands separate forming a replication fork (bidirectional) -each strand is replicated by complementary base pairing -replication is semiconservative -helicases: open unwind and stabilize strands -DNA polymerase: synthesize strand in 5' to 3' direction, needs primer to begin -Leading strand: forms continue 5' to 3' -Laggin Strand: forms in okazaki fragments 5' to 3' -ligase joins fragments (primer needed)
Types of Mutations and their effects
-before we can consider mutations and their effects, we need to distinguish between an organisms genotype and phenotype -genotype: referes to the genetic information contained in the DNA of the organism -Phenotype: refers to the specific characteristics displayed by the organism -mutations always change the genotype -such a change may or may not be expressed in the phenotype, depending on the nature of the mutation -two important kinds of mutations are point mutations, which affect a single base, and frameshift mutations, which can affect more than one base in DNA -mutations often make an organism unable to synthesize one or more proteins -the absence of a protein often leads to changes in the organisms structure or in its ability to metabolize a particular substance -a third type of mutation does not involve a change as to which bases are present, as is the case in point and frameshift mutations -instead, a portion of the chromosome changes its position, perhaps even breaking off and jumping to another part of the same or a different chromosome (transposons) -or it may reinsert itself in the same location, but upside down (inversions) -as you think back to the lac operon, you can see why it is important for genes to retain their correct order on a chromosome -imagine what would happen if a piece of chromosome suddenly inserted itself into the middle of an operon
Transposable elements
-can cause mutations -Transposons-DNA segments that move within genome
Point Mutations
-change in one base pair -base substitution:during replication -Missense: altered protein produced -Nonsense: no protein produced -Single base change in DNA with no change in the amino acid specified by the mRNA codon: no effect on protein, a "silent" mutation -Change in DNA with change in the amino acid sequence specified by the mRNA codon: change in protein by substitution of one amino acid for another; can significantly alter function of a protein -Change in DNA that creates a terminator codon in mRNA: produces polypeptide of no use to organism and prevents synthesis of normal protein
A typical prokaryotic cell...
-contains a single circular chromosome, composed primarily of a single DNA molecule about 1 mm long when fully stretched out--some 1,000 times longer than the cell itself -this immense molecule fits compactly into the cell, where it forms the nucleoid by twisting tightly around itself, a process known as supercoiling -when a prokaryotic cell reproduces by binary fission, the chromosome reproduces, or replicates itself, and each daughter cell receives one of the chromosomes. -this mechanism provides for the orderly transmission of genetic information from parent cell to daughter cells
Information Transfer
-information stored in DNA is used both to guide the replication of DNA in preparation for cell division and to direct protein synthesis -the three ways in which this information is transferred are as follows: --replication: DNA makes new DNA --Transcription: DNA makes RNA as the first step in protein synthesis --Translation: RNA links amino acids together to form proteins -in both DNA replication and transcription, DNA serves as a template for the synthesis of a new nucleotide polymer -the sequence of bases in each new polymer is complementary to that in the original DNA -such an arrangement is accomplished by base pairing -recall that when DNA serves as a template for synthesis of RNA, the pairing is different: in RNA, thymine is replaced by uracil, with pairs with adenine
Chromosomes
-linear in eukaryotes -circular in prokaryotes -Gene: sequence of DNA, defines a characteristic -Alleles: alternate form of a gene -Prokaryotes: one chromosome, one allele to a gene -Eukaryotes: pairs of chromosomes, 2 alleles to a gene -Mutation: a permanent alteration in DNA -mutations usually change the sequence of nucleotides in DNA and thereby change the information in the DNA -when the mutated DNA is transmitted to a daughter cell, the daughter cell can differ from the parent cell in one or more characteristics -heritable variations in the characteristics of progeny can arise from mutations
Polyribosome
-many ribosomes translate same mRNA -Translation can begin before transcription is finished in prokaryotes but not in eukaryotes -concurrent transcription and translation in prokaryotes -the presence of many ribosomes, all "riding" simultaneously along one piece of mRNA, give this name polyribosome
Mutations
-mutations or changes in DNA, can now be defined more precisely as heritable changes in the sequence of nucleotides in DNA -Mutations account for evolutionary changes in microorganisms (and larger organisms) and for alterations that produce different strains within species -here we will consider how DNA changes during mutations and how these changes affect the organisms
Spontaneous and Induced Mutations
-occur in the absence of any agent known to cause changes in DNA -they arise during the replication of DNA and appear to be due to errors in the base pairing of nucleotides in the old and new strands of DNA -various genes in the DNA of bacteria have different spontaneous mutation rates, ranging from 10^-3 to 10^-9 per cell division -in other words, one gene might undergo a spontaneous mutation once in every thousand cell divisions, where as another gene might undergo spontaneous mutation only once in every billion cell divisions. -random, during DNA replication, error in base pairing--base substitution (point mutation) -rate of mutation: probability that a mutation will occur every time a cell divides
Natural Selection
-organism with mutation selected by the environment--survives, becomes dominant -evolution of microorganisms -it is important to differentiate between spontaneous and induced mutations -resistance to penicillin due to spontaneous mutation which the environment can select -it is not caused by penicillin -resistance arises from spontaneous random mutations : organism with mutation selected by the environment - survives, becomes dominant. Evolution of microorganisms It is important to differentiate between spontaneous and induced mutations. • Helps us understand mechanism in the evolution of microorganisms and presumably other organisms as well. Resistance to penicillin due to a spontaneous mutation which the environment can select. It is not caused by the penicillin. Resistance arises from spontaneous random mutations.
Induced Mutations
-produced by agents called mutagens, which increase the mutation rate above spontaneous mutation rate -mutagens include chemical agents and radiation -caused by chemical or physical agents
Translation
-protein synthesis, is an important process in bacterial growth, uses 80 to 90% of a bacterial cell's energy -generally, during protein synthesis, the various RNAs and amino acids are available in sufficient quantities -the RNAs can be reused many times before they lose their ability to function -of the types of RNA, mRNA is produced in the most precise quantity in accordance with the cell's need for a particular protein -three types of RNA and how they function in protein synthesis ----1.) rRNA complexes with proteins to form the ribosomes ----2.) on the ribosomes, the mRNA message is read and the proteins are assembled ----3.) tRNA molecules carry amino acids to the ribosomes to be incorporated into proteins -once an mRNA molecule has been transcribed and has combined with a ribosome, the ribosome initiates protein synthesis and provides the site for protein assembly -each ribosome attaches first to the end of the mRNA that corresponds to the beginning of a protein -the length of each polypeptide chain extending from a ribosome corresponds to the amount of mRNA the ribosome has "read" -several ribosomes can be be attached at different points along an mRNA molecule to form a polyribosome -in prokaryotes, transcription and translation take place in the cytoplasm, where all necessary enzymes and ribosomes are present -in eukaryotes, the mRNA formed in the nucleus must pass through the nuclear membrane before it is available to the ribosomes, which carry out protein synthesis -the main steps in protein synthesis can be summarized as follows --the process begins when a molecule of mRNA becomes properly oriented on a ribosome --as each codon of the mRNA is "read", the appropriate tRNA combines with it and thereby delivers a particular amino acid to the protein assembly site. --the location on the ribosome where the first tRNA pairs is called the P site. --the second codon of the mRNA then pairs with a tRNA that transports the second amino acid to the A site, which is next to the P site --Matching of codon and anticodon by base pairing allows coded information in mRNA to specify the sequence of amino acids in a protein --any tRNAs with non matching anticodons simply do not bind to the ribosome --as amino acids are delivered one after another and peptide bonds form between them, the length of the polypeptide chain increases --this process continues until the ribosome recognizes a stop codon --when the ribosome "reads" a stop codon at the A site, it releases the finished protein from the P site -any mRNA molecule can direct simultaneous synthesis of many identical protein molecules--one for each ribosome passing along it -ribosomes, mRNAs, and tRNAs are reusable -the tRNAs shuttle back and forth picking up amino acids in the cytoplasm, and bringing them to the ribosome, where the amino acids are incorporated into protein
DNA Replication
-to understand DNA replication, we need to understand a couple things -the ends of each strand is different -at one end, called the 3' end, carbon 3 deoxyribose is free to bind to other molecules -at the other end, the 5' end, carbon 5 deoxyribose is attached to a phosphate -this structure is somewhat analogous to that of a freight train, with the 3' end the engine and the 5' end the caboose. -when the two strands of a double helix combine by base pairing, they do so in a head to tail, or antiparallel, fashion -the arrangement of the strands is somewhat like two trains pointed in opposite direction, and base pairing is like passengers in the two trains shaking hands -DNA replication begins at a specific location (the origin) in the circular chromosome of a prokaryotic cell and usually proceeds simultaneously away from the origin in both directions -this creates two moving replication forks, the points at which the two strands of DNA separate to allow replication of DNA -various enzymes (helicases) breaks the hydrogen bonds between the bases in the two DNA strands, unwind the strands from each other, and stabilize the exposed single strands, preventing them from joining back together -molecules of the enzyme DNA polymerase then move along behind each replication fork, synthesizing new DNA strands complementary to the original ones at a speed of approximately 1000 nucleotides per second -DNA polymerase also "proofreads" the growing strand, correcting errors such as mismatched bases -even at such high speeds, proofreading usually leaves only one in 10 base pairs with an error -the enzyme DNA polymerase can add nucleotides only to the 3' end of a growing DNA strand -consequently, only one strand of original DNA can serve as a template for the synthesis of a continuous new strand, the leading strand, going in the 5' to 3' direction -along the other strand, which runs in the 3' to 5' direction, the synthesis of new DNA, the lagging strand, must be discontinuous; that is, the polymerase must continually jump ahead and work backward, making a series of short DNA segments called okazaki fragments, which consists of 100 to 1000 base pairs -each fragment must have a short piece of RNA called an RNA primer attached to the parent DNA in order to start synthesis of new DNA -Later DNA polymerase will digest the RNA primer and replace it with DNA -the fragments are then joined together by another enzyme called ligase -formation of leading and lagging strands goes on simultaneosly -but because the DNA polymerase producing okazaki fragments must wait until enough DNA has been opened up at the replication fork for an RNA primer to form, it is said to be "lagging" -ultimately, two separate chromosomes are formed, each double helix consisting of one of old, or parent, DNA and one strand of new DNA -such replication is called semiconservative replication because one strand is always conserved
Testing for Carcinogens
Ames Test: • Based on the ability of auxotrophic bacteria to mutate by reverting to their original synthetic ability. It is used by screening chemicals for mutagenic properties, which indicate potential carcinogens. • Carcinogens: cancer producing compounds • Used to test whether substances induce mutations in certain strains of salmonella (auxotrophs) that have lost their ability to synthesize histidine. The amest test is based on the hypothesis that if a substance is a mutagen, it will increase the rate at which these organisms revert to being histidine synthesizers. • The more powerful a substances mutagenic capacity, the greater the number of reverted organisms it causes to appear. • If any organisms regain the ability to synthesize histidine, the substance is suspected of being a mutagen. • The larger the number of organisms that regain the synthetic ability, the stronger the substance's mutagenic capacity is likely to be.
Some mutagens and their effects
Base Analog (Caffeine, 5-bromouracil): substitutes "look-alike" molecule for the normal nitrogenous base during DNA replication--> point mutation Alkylating Agents (nitrosoguanidine): adds an alkyl group, such as a methyl group (-CH3), to a nitrogenous base, resulting in incorrect pairing --> mutation -mustard gas Deaminating Agent (nitrous acid, nitrates, nitrites): removes an amino group (-NH2) from a nitrogenous base --> point mutation -nitrous oxide Acridine derivative (acridine dyes, quinacrine): inserts into DNA ladder between backbones to form a new rung, distorting the helix--> frameshift mutation -ethidium bromide
Protein Synthesis
DNA transcription, mRNA translation, polypeptide (protein)
Tests for Isolating mutants
Direct Selection: Fluctuation Test- is based on the following hypothesis: if mutations that confer resistance occur spontaneously and at random, we would expect great fluctuation in the number of resistant organisms per culture among a large number of cultures -this fluctuation would occur regardless of whether the substance to which resistance develops is present • Based on the following hypothesis • If mutations that confer resistance occur spontaneously and at random, we would expect great fluctuation in the number of resistant organisms per culture among a large number of cultures. • Demonstrates that resistance to chemical substances occurs spontaneously rather than being induced Indirect Selection: Replica Plating • Demonstrates the spontaneous nature of mutations; it also can be used for isolating mutants without exposing them to a substance to which they are resistant • Used to study mutations • It hypothesizes that resistance to a substance arises spontaneously and at random without the need for exposure to the substance
Types of Mutations
Point Mutations Frameshift Mutations Transposable Elements
Repair of DNA damage
Proofreading: by DNA polymerase Mismatch repair: by enzyme system-endonuclease cut excise mistake, DNA polymerase fill gap, DNA ligase joins strand -many bacteria, and other organisms as well, have enzymes that can repair certain kinds of damage to DNA -two mechanisms, light repair and dark repair, are known to repair damage caused by dimers Light repair or photo reactivation: occurs in the presence of visible light in bacteria previously exposed to UV light -when organisms containing dimers are kept in visible light, the light activates an enzyme that breaks the bonds between the pyrimidines of a dimer -thus mutations that might have been passed along to daughter cells are corrected, and the DNA is returned to its normal state -this mechanism contributes to the survival of the bacteria but creates a problem for microbiologists -cultures that are irradiated with UV light to induce mutations must be kept in the dark for mutations to be retained. -photoreactivation: in presence of visible light enzyme breaks bond between thymines Dark repair: occurs in some bacteria, and can take place in the presence or absence of light, requires several enzyme controlled reactions -first, an endonuclease breaks the defective DNA strand near the dimer -second, a DNA polymerase synthesizes new DNA to replace the defective segment, using the normal complementary strand as a template -third, an exonuclease removes the defective DNA segment -finally, a ligase connects the repaired segment to the remainder of the DNA strand -these reactions were identified in E.coli but are now known to occur in many other bacteria -human cells have similar mechanisms, some human skin cancers, such as xeroderma pigmentosum, are caused by a defect in the cellular DNA repair mechanism -enzyme excise and repair damage -Endonuclease cuts strand, DNA polymerase replaces damage, exonuclease removes damaged segment, ligase connects repaired segment to strand
Transcription (figure 7.5)
RNA: single strand; uracil substitutes for thymine; synthesis by complementary base pairing in 5' to 3' direction -DNA opens, one strand used as a template -RNA polymerase binds to promoter and transcribes gene: initiation, elongation, termination-RNA released -prokaryotes: transcription and translation in cytoplasm -eukaryotes: transcription in nucleus, translation in cytoplasm -processing of mRNA: introns removed, exons spliced--continuous mRNA (gene) -Cap of GTP at 5', Poly A tail at 3' -mRNA exits nucleus
Kinds of RNA
Ribosomal: -combines with specific proteins to form ribosomes -serves as a site for protein synthesis -associated enzymes function in controlling protein synthesis Messenger: -carries information from DNA for synthesis of a protein -molecules correspond in length to one or more genes in DNA -has base triplets called codons that constitute the genetic code -attaches to one or more ribosomes Transfer: -Found in the cytoplasm, where they pick up amino acids and transfer them to mRNA -molecules have a cloverleaf shape with an attachment site for a specific amino acid -each has a single triplet of bases called an anticodon, which pairs complementary with the corresponding codon in mRNA -brings proper amino acid to ribosomes for protein synthesis -many tRNAs -site for amino acid attachment -Anticodon: complementary to codon in mRNA -Complementary base pairing during polypeptide synthesis READ THESE IN THE BOOK***
Radiation as a Mutagen
Ultraviolet: links adjacent pyrimidines to each other, as in thymine dimer formation, and thereby impairs replication -thmine dimers, produce gap in replicated DNA, prevent transcription -dimer: consists of two adjacent pyrimidines bonded together in a DNA strand X-ray and gamma ray: ionize and break molecules in cells to form free radicals, which in turn break DNA -break DNA Restriction endonucleases: cut DNA at precise base sequences Exonucleases: remove segments of DNA -these enzymes allow individual genes to be isolated and mutated at predetermined sites -the mutated gene can be inserted into a hosts chromosome and the effect of the specific mutation studied.
Enzyme Induction
o In enzyme induction, the presence of a substrate activates an operon, a sequence of closely associated genes that includes structural genes and regulatory sites ♣ In the absence of lactose, a repressor—a product of the regulator gene—attaches to the operator and prevents transcription of the genes of the lac operon. ♣ When lactose is present, it inactivates the repressor and allows transcription of the genes of the lac operon.
Enzyme Repression
o In enzyme repression, the presence of a synthetic product inhibits its further synthesis by inactivating an operon ♣ When tryptophan is present, it attaches to the repressor protein and represses genes of the trp operon ♣ In the absence of tryptophan, the repressor is not activated, and the genes of the trp operon are transcribed o In catabolic repression, the presence of a preferred nutrient (often glucose) represses the synthesis of enzymes that would be used to metabolize some alternative substances. o Both enzyme induction and enzyme repression regulate by altering gene expression. The effect on enzyme synthesis in both cases depends on the presence or absence of the regulatory substance—lactose, tryptophan, or glucose in the preceding examples.
Feedback Inhibition
o In feedback inhibiton, the end product of a biochemical pathway directly inhibits the first enzyme in the pathway o Enzymes subject to such regulation are generally allosteric o Feedback inhibition regulates the activity of existing enzymes and is a quick acting control mechanism
Significance
o Mechanisms that regulate metabolism turn reactions on and off in accordance with the needs of the cells, allowing the cells to use various energy sources and to limit synthesis of substances to the amounts needed.
Categories of Regulatory Mechanisms
o The two basic categories of regulatory mechanisms are ♣ Mechanisms that regulate the activity of enzymes already available in the cell ♣ Mechanisms that regulate the action of genes, which determine what enzymes and other proteins will be available
Gene
the basic unit of heredity, is a linear sequence of nucleotides of DNA that forms a functional unit of a chromosome or plasmid -all information for the structure and function of an organism is coded in its genes -in many cases, a gene determines a single characteristic -however, the information in a specific gene, found at a particular locus(location) on the chromosome or plasmid, is not always the same -genes with different information at the same locus are called alleles -because prokaryotes have a single chromosome, they generally have only one version, or allele of each gene -many but not all eukaryotes have two sets of chromosomes and thus two alleles of each gene, which may be the same or different -for example, in human blood types, and one of three gene variants, or alleles--A, B, or O--can occupy a certain locus -Allele A causes red blood cells to have a certain glycoprotein, which we will designate as molecule A, on their surfaces -Allele B causes them to have molecule B -Allele O does not cause them to have any glycoprotein molecule on the cell surfaces -people with try AB blood produce both molecules A and B because they have alleles A and B
