BIO 245 Ch 10 & 11 PDF

Chapter 10 - Biochemistry of the Genome mRNA- carries the message from DNA to make a protein ○ Single stranded, complementary copy of DNA template ○ Synthesized through transcription If a cell required a certain protein to be synthesized - gene for the...

Chapter 10 - Biochemistry of the Genome mRNA- carries the message from DNA to make a protein ○ Single stranded, complementary copy of DNA template ○ Synthesized through transcription If a cell required a certain protein to be synthesized - gene for the product is turned on ○ Located in nucleus (eukaryotic cells) or cytoplasm (prokaryotic cells) ○ Directs protein synthesis, called translation Interacts with ribosomes ○ Relatively unstable and short lived rRNA- has structural and catalytic role ○ Stable ○ Synthesized in nucleolus (eukaryotic) or cytoplasm (prokaryotic) ○ Makes up 60% of a ribosome ○ During translation - ensures proper alignment of the mRNA, tRNA, and ribosomes ○ Catalyzes formation of peptide bonds with peptidyl transferase tRNA ○ Stable ○ Smallest type of RNA (only 70-90 nucleotides long) ○ Carries correct amino acid to site of protein synthesis in ribosome Codon (mRNA) or anticodon (tRNA) ○ Intracellular base pairing gives it its characteristic shape 2D - clover shape 3D - L shape RNA does not carry hereditary info ○ Exception is some viruses or prehistoric Genome- all of an organism’s genetic material ○ Includes chromosomes and plasmids Chromosomes- structures containing DNA that physically carry genetic information ○ Chromosomes contain genes Genes- segments of DNA that encode functional products, usually proteins Genotype- the genetic makeup of an organism ○ Full collection of genes that a cell contains within its genome Phenotype- expression of the genes, characteristics that are visible Eukaryotic chromosome ○ Linear ○ Multiple distinct chromosomes  ○ Diploid- two copies of each chromosome ○ Length- much longer than cell DNA supercoiling- twisting of DNA to fit within cell Topoisomerases- enzymes that prevent supercoiling ○ Histones- DNA binding proteins that DNA wraps around for protein attachment Chromatin- thread of DNA and attached histones Packaging influenced by environmental factors DNA methylation Epigenetics Prokaryotic chromosome ○ Circular ○ Single chromosome in nucleoid ○ Haploid- one copy of each chromosome ○ Length- much longer than cell (like eukaryotes) DNA supercoiling by topoisomerase called DNA gyrase Histone-like proteins present Extrachromosomal DNA ○ DNA external of chromosome but part of genome ○ Mitochondria and chloroplast chromosomes are circular in eukaryotes ○ DNA latent viruses in host cells ○ Plasmids Not essential for cellular growth Involved in horizontal gene transfer Recall griffith’s experiment Utilized in genetic transfer research and biotechnology Chapter 11 - Mechanisms of Microbial Genetics Functions of DNA ○ 1) responsible for inheritance Vertical transfer- passed from parent to offspring Through DNA replication with minimal errors ○ 2) direct and regulate protein synthesis Required for cell growth and reproduction in a particular environment One gene one enzyme hypothesis Central dogma: DNA → RNA → Protein ○ Transcription- info from DNA is transferred to RNA (mRNA) ○ Translation- info from mRNA is used to build polypeptide proteins  DNA replication- double helix is uncoiled and separated into single strands Proposed models ○ Conservative ○ Semiconservative ○ Dispersive DNA replication is semiconservative- one strand is conserved and one is newly replicated ○ Each single strand served as a template for new complementary strand ○ After replication, each double stranded DNA includes one “old” strand and one “new” strand DNA replication in bacteria ○ Initiation Occurs at origin of replication- specific nucleotide sequence oriC for prokaryotes Topoisomerase II (DNA gyrase)- relaxes supercoiled DNA strands Helicase- unzips DNA strands by breaking hydrogen bonds A and T have 2 H bonds G and C have 3 H bonds Y shaped replication fork is created Two replication forks allow bidirectional replication Replication bubble formed Single stranded binding proteins- prevent single stranded DNA from rejoining ○ Replication begins DNA polymerase III adds nucleotides to growing DNA strand Always synthesized in 5’ → 3’ direction Can only add a nucleotide onto a preexisting 3’ OH group Cannot make things from scratch RNA primer- initiates replication by providing free 3’- OH end Complementary to template DNA RNA primase- synthesizes RNA molecule Does not need a free 3’- OH end to synthesize RNA ○ Elongation Addition of nucleotides Adds 1000 nucleotides per second Sliding clamp- holds DNA polymerase III in place Leading strand is synthesized continuously Requires one primer Lagging strand is synthesized discontinuously, creating Okazaki fragments  Requires a new primer for each Okazaki fragment DNA polymerase I- removes RNA primers DNA ligases- seals gaps ○ Termination Not much is known about it In prokaryotes, circular genomes are concatenated (=interlocked) Topoisomerase IV reseals the chromosomes Eukaryotic genomes ○ Much more complex and larger than prokaryotic genome ○ Composed of multiple linear chromosomes ○ Multiple origins of replication Fast rate of replication 100 nucleotides per second (prokaryotes can do 1000 per second) ○ Similar steps (initiation, elongation, termination) Elongation in eukaryotes- facilitated by DNA polymerase ○ Lead strand- synthesized continuously by enzyme pol epsilon 𝝴 ○ Lagging strand- synthesized discontinuously by pol delta 𝝳 ○ Ribonuclease H (RNase H) removes RNA primer ○ Ligase seals the gaps property bacteria eukaryotes Genome structure Single circular chromosome Multiple linear chromosomes Origins per chromosome one multiple Rate of replication 1000 nucleotides/second 100 nucleotides/second RNA primer removal DNA pol I RNase H Strand elongation DNA pol III Leading: pol 𝝴 Lagging: pol 𝝳 RNA transcription- process of copying info encoded into DNA into a strand of RNA ○ One DNA strand serves as template  ○ Result is a RNA transcript Complementary to template strand U swaps with T, almost identical to non template strand ○ RNA polymerase transcribes in 5’ to 3’ direction ○ DNA double helix must partially unwind & form transcription bubble Transcription in bacteria (3 steps) ○ Only one RNA polymerase ○ Five subunits compose polymerase core enzyme Adds RNA nucleotides ○ 6th subunit is sigma factor Directs RNA pol binding to specific promoter ○ Adds nucleotides to 3’-OH group to form phosphodiester bond ○ Does not require RNA primer Initiation ○ Transcription begins when RNA polymerase binds to the promoter sequence on DNA ○ Initiation (start) site- pair of nucleotides in double helix designated +1 Nucleotides preceding initiation site are called upstream Nucleotides following initiation site are called downstream Elongation ○ Begins when sigma factor dissociates from RNA polymerase ○ Allows core enzyme to synthesize RNA complementary to the DNA template In 5’ to 3’ direction 40 nucleotides per second ○ RNA polymerase DNA is unwound ahead of core enzyme Rewound behind it Termination ○ Dissociation of RNA pol from DNA template Newly formed RNA is released Signaled by repeated nucleotide sequences ○ Influenced by Rho protein (= Rho dependent termination) ○ Influenced by RNA hairpin (stem loop) formation (=Rho INDEPENDENT termination) Transcription in Eukaryotes ○ Uses 3 different RNA polymerases archaea - uses one RNA pol (like bacteria) but more closely related to Euk RNA pol II  ○ Eukaryotic mRNAs are monocistronic (encode only a single polypeptide) ○ Prokaryotic mRNAs are polycistronic (encode multiple polypeptides) ○ pre-mRNA is processed before transport to cytoplasm Addition of 5’ cap Prevents degradation Recognized by translational factors Addition of 3’ poly A tail String of 200 Adenine nucleotides Further protects against degradation ○ RNA splicing- removal of introns and rejoining of exons Facilitated by spliceosome made of snRNPs (snurps) Introns (intervening)- regions of DNA that do NOT code for proteins Exons (expressed)- regions of DNA that code for proteins Translation (protein synthesis) ○ mRNA is translated into language of amino acids by ribosomes ○ Genetic code- relationship between an mRNA codon and its corresponding amino acid ○ Codons- groups of 3 amino acids that code for a particular amino acid 64 possible codons (4^3) Only 20 amino acids ○ Degeneracy (redundancy) of genetic code- the same amino acid can be coded by several codons ○ 3rd position (wobble position) - can be different and the same amino acid will still be produce ○ Start codon: AUG Translation begins Determines the reading frame Also codes for Met ○ 61 sense codons encode 20 amino acids ○ 3 nonsense codons (STOP codons) UAA, UAG, UGA terminate translation, polypeptide is released Protein synthesis machinery ○ mRNA template ○ tRNAs ○ Various enzymatic factors  ○ Ribosomes- site of translation Prokaryotes- 30S + 50S makes 70S Eukaryotes- 40S + 60S makes 80S Small subunit binds the mRNA template Large subunit binds tRNAs ○ Prokaryotes- transcription and translation occurs in cytoplasm Translation can begin before transcription is complete ○ Polyribosomes (polysomes)- complex formed by ribosomes simultaneously translating mRNA tRNAs in translation ○ tRNAs- translate the language of RNA into proteins Transports required amino acids to the ribosome Cloverleaf shape ○ Anticodon- 3 bases of tRNA that recognize 3 complementary bases (codon) on mRNA ○ Amino acid attachment site- amino acid corresponding to mRNA codon is attached Charged tRNA is formed when amino acid attaches to a tRNA Translation steps ○ Initiation: Formation of initiation complex Small 30S ribosome subunit mRNA template Special initiator tRNA carrying n-formylmethionine (or Methionine in eukaryotes) Large 50S subunit binds, forming an intact ribosome Begins at start codon: Prokaryotes have Shine-dalgarno sequence- base pairing between rRNA and mRNA template Eukaryotes - no SD sequence - involves 5’ cap and 3’ poly A tail ○ Elongation Intact ribosome has 3 sites: Aminoacyl (A) site- binds incoming charged aminoacyl tRNAs Peptidyl (P) site- binds charged amino acid associated tRNAs with peptide bonds ○ Initiator tRNA binds at P site, the rest of the aminoacyl tRNAs bind in A site  ○ Peptide bond formed between A site amino group and P site carboxyl group Exit (E) site- dissociated tRNAs released for recharging Translocation- single codon ribosomal movements (sequence of 3) Charged tRNAs shift from A→P→E Termination ○ Alignment of A site with nonsense codons (STOP codons) ○ Released by release factors P site amino acid is attached from tRNA Newly made polypeptide is released ○ Dissociation of ribosomal subunits Components recycled property bacteria eukaryotes ribosomes 70S 80S 30 S and 50S 40S and 60s Amino acid carried by fMet Met initiator tRNA Shine-dalgarno sequence in yes no, instead has 5’ cap and mRNA poly A tail Simultaneous transcription yes no and translation Mutation- heritable change in the DNA sequence of an organism ○ Produces a mutant May have a recognizable change in phenotype Change in amino acid sequence in protein Wild type- phenotype that is most commonly observed ○ Mutations can be neutral, beneficial, or harmful ○ Mutagens- agents that cause mutations ○ Spontaneous mutations- occur in the absence of a mutagen Types of mutations ○ Point mutation- affects a single base (base substitution) ○ Frameshift mutation- insertion/deletion of one or more nucleotides Shifts the reading frame, leads to different amino acid sequence Insertion mutation- addition of one or more bases  Deletion mutation- removal of one or more bases ○ If in groups of three nucleotides, may not cause significant effects on protein’s functionality Effects of point mutations ○ Silent mutation- no change in AA Due to degeneracy of genetic code Has no effect on the protein’s structure Ex: GUA becomes GUU, they're both valine ○ Missense mutation- change in AA Rarely beneficial Protein usually maintains function Ex: CCC proline becomes ACC threonine ○ Nonsense mutation- results in a nonsense (STOP) codon Proteins are shorter and not functional Worst type of the 3 Ex: UAC tyrosine becomes UAG STOP codon Causes of mutations ○ Very rare ○ Spontaneous mutation- due to errors made by DNA pol during replication ○ Mutation rate- probability that a gene will mutate when a cell divides One in a billion replicated base pairs One in a million replicated genes ○ Mutagens increase mutation rate by 1000 per replicated genes Can also be carcinogenic Types of mutagens ○ Chemical: Nitrous acid (HNO2)- modifies normal DNA bases Deaminates cytosine converting it to uracil ○ Uracil then pairs with adenine ○ Results in conversion of GC base pair to AT Nucleoside analogs- structurally similar to normal nucleotide bases Can be incorporated into DNA during replication Causes mistakes in base pairing Intercalating agents- slide between the stacked bases of DNA double helix Distorts DNA, can lead to frameshift mutation ○ Radiation:  Ionizing radiation (x rays and gamma rays)- causes formation of hydroxyl radicals Produces single and double stranded breaks in DNA backbone Non Ionizing radiation (UV rays)- induce dimer formation between two adjacent pyrimidine bases Thymine dimers- two adjacent thymines become covalently linked ○ Both DNA replication and transcription are stalled ○ Leads to frameshift mutations How asexual prokaryotes achieve genetic diversity ○ Vertical gene transfer- transmission of genetic information from generation to generation Sexual reproduction - crossing over, independent assortment, and mutations contribute to genetic diversity Asexual reproduction - nearly identical copy of genetic material passed to offspring, more offspring produced more quickly, diversity among offspring is lost ○ In asexual reproduction → genetic diversity achieved through horizontal gene transfer (= introduction of genetic material from one organism to another organism within the same generation) Transformation, transduction, conjugation Transformation- genes transferred from one bacterium to another as naked DNA ○ Recall griffith’s experiment ○ After death, some bacteria release DNA into environment Other bacteria may pick up these pieces of DNA Integrates into chromosome by recombination Recombinant DNA- acquire new phenotypic properties ○ Toxin production, antibiotic resistance ○ Plasmid DNA can also be picked up ○ Competence- ability of a bacteria to take in donor DNA Naturally competent bacteria- Bacillus, Haemophilus, Acinetobacter Transduction- DNA is transferred from a donor cell to a recipient via a bacteriophage (=virus that infects bacteria) ○ Generalized transduction- random bacterial DNA is packaged inside a phage and transferred to a recipient cell - lytic cycle ○ Specialized transduction- specific bacterial genes are packaged inside a phage and transferred to a recipient cell - lysogenic cycle  Conjugation- DNA is directly transferred from one prokaryote to another by means of a conjugation pilus ○ Requires cell-to-cell contact ○ Typically involved transfer of F plasmids F plasmid- fertility factor Conjugation pilus is called F pilus ○ Cells containing F plasmid are called F+ cells/donor cells ○ Cells lacking F plasmic are called F- cells/recipient cells ○ F pilus of an F+ cell comes into contact with F- cell Retracts, bringing the two cell envelopes into contact A cytoplasmic bridge forms between the two cells One strand of the F plasmid is transferred F- cell becomes an F+ cell capable of making its own conjugation pilus ○ If the F plasmid integrates into the chromosome, the cell becomes an Hfr cell (high frequency of recombination) Transposition ○ Transposons- jumping genes Special inverted terminal repeat sequences Gene encoding transposase ○ Transposons can independently excise and integrate into a new DNA location (=transposition) Usually nonreplicative- move in a cut and paste fashion Can be replicative- copy and paste fashion ○ Found in both eukaryotes and prokaryotes ○ Can introduce genetic diversity and alter phenotype Promote transfer of antibiotic resistance genes (from chromosomes to plasmids) Gene regulation ○ Gene expression- highly regulated process Multicellular organisms- for cellular differentiation Single celled organisms (prokaryotes)- primarily ensures that a cell’s resources are not wasted making proteins that the cell does not need at that time Operon model of gene expression ○ Promoter- segment of DNA where RNA polymerase initiates transcription of structural genes ○ Operator- segment of DNA that controls transcription of structural genes ○ Structural genes- encodes enzymes needed  ○ Operon- set of promoter and operator sites and the structural genes they control ○ Promoter and operator also involves a regulatory gene Prokaryotic gene regulation ○ Controlled by a single promoter- produces a polycistronic transcript ○ Influenced by binding of transcription factors (TF) or small molecules Repressor- TF that suppresses transcription Binds to operator- blocks RNA polymerase binding to promoter Activator- TF that enhances transcription Facilitates RNA polymerase binding to promoter Inducer- small molecule that activates or represses transcription via interaction with an activator or repressor Inducible operon (lac operon) ○ Lac operon- encodes three structural genes necessary to breakdown lactose into glucose and galactose lac Z - beta galactosidase ○ Absence of lactose = repressor binds to the operator, preventing transcription ○ Lactose in excess = converted into allolactose Allolactose is an inducer Binds to the repressor, then the repressor cannot bind to operator and transcription occurs Repressible operon (trp operon) ○ trp operon- for synthesis of tryptophan Using enzymes that are encoded by five structural genes located next to each other ○ Absence of tryptophan = repressor doesn’t bind to operator RNA polymerase binds instead, transcription is initiated and tryptophan is synthesized ○ Tryptophan in excess is a corepressor Binds and activates the repressor to bind to the operator Stops tryptophan synthesis Catabolite repression ○ Bacteria can utilize a variety of substrates as carbon sources Glucose is preferred If glucose is depleted → lactose is used ○ If both glucose and lactose present: Uses glucose first, grows fast Once glucose is depleted, growth rates slow  Induces expression of the enzymes needed for metabolism of glucose Starts growing again but at a slower rate