Summary

These notes cover the biochemistry of the genome and mechanisms of microbial genetics, including details on mRNA, rRNA, tRNA, DNA replication, and transcription. Excellent study material for BIO 245.

Full Transcript

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 tur...

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 re-joining 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

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