Central Dogma of Biology.pdf

LG 2.1 Roles and Components of Nucleic Acids Topic Summary Historical Overview Avery, McCarty, MacLeod Search for genetic material done by Oswald Avery, Maclyn McCarty and Colin MacLeaod...

LG 2.1 Roles and Components of Nucleic Acids Topic Summary Historical Overview Avery, McCarty, MacLeod Search for genetic material done by Oswald Avery, Maclyn McCarty and Colin MacLeaod ○ Purified the transforming principle ○ Exposed it to different enzymes to see reaction Hershey and Chase Scientists also used bacteriophages → Viruses that infect bacteria Alfred Hershey and Martha Chase ○ One batch with radioactive sulfur isotope that labeled phage proteins ○ One batch with radioactive phosphate isotope that labeled phage DNA Supported that DNA transferred the bacteria Rosalind Franklin Rosalind Franklin moved to Paris and perfected x-ray crystallography technique ○ Returned to London and worked with Maurice Wilkins to find DNA structure ○ Conflict occurred and Wilkins shared Franklin’s data to scientists James Watson and Francis Crick ○ Franklin’s Photo 51 Structure for Watson and Crick published work characterizing DNA structure Deoxyribonucleic Acid Double helix illustrated by Odile, Crick’s wife Distinct pairing of nitrogenous bases on the inside of two sugar-phosphate backbones ○ Adenine=Thymine and Cytosine=Guanine Distance between base pairs is ladder-like Double helix model is 0.34 nm and 10 base pairs in one full turn ○ Presence of two strands composed of sugar-phosphate backbone Sugar-Phosphate Backbone Comprises periphery of the helix  Adenine and Thymine bound by two hydrogen bonds Cytosine and Guanine bound by three hydrogen bonds LG 2.2 Overview of the Molecular Dogma Topic Summary Nucleic Acids Nucleic acids → molecules transmitting biological info from parent to daughter cell One generation to another Made of nucleotides → made of sugar-phosphate backbone and nitrogenous base Nitrogenous Base May be purine of a pyrimidine Attached to first carbon of pentose sugar Phosphate group attached to fifth carbon (5’ end or 5’ phosphate) Hydroxyl group attached to third carbon (3’end or 3’ OH) ○ Where next 5’-phosphate of succeeding nucleotide will attach to DNA and RNA DNA is double stranded → opposite directions, RNA is single stranded Both have pentose-phosphate backbones and nitrogenous bases Sugar backbone for DNA is deoxyribose, RNA is ribose Thymine is unique to DNA; uracil is unique to RNA Central Dogma DNA replication (DNA synthesis), transcription (synthesis of RNA), and translation (protein synthesis) DNA Semi-conservative replication → new DNA contains old strand from parent and newly formed strand Two parent strands are templates Complementary strands are synthesized Accuracy ensured by specific base pairing and enzyme proofreading DNA Replication Initiation Separation of complementary strands to be templates Helicase protein/enzyme unwinds two strands by breaking hydrogen bonds Single stranded binding proteins bind to single strand to prevent them from getting back together Topoisomerases relaxes twisting tension created Short RNA sequences are synthesized by primase → primers for elongation ○ Completes DNA with short stretches of RNA in 5’ to 3’ direction ○ Provide 3’-OH group needed for phosphodiester bonds for nucleotides added in elongation Distinctive fork-like structure that exposes nitrogenous bases of template strand Elongation DNA Polymerase III adds triphosphates complementary to DNA template that starts from 3’-OH end of primer Complement strand elongates in same direction of unwinding fork → leading strand ○ Requires only one primer and is 5’ to 3’ in direction Other strand is replicated in opposite direction of replication fork → lagging strand ○ 3’ to 5’ direction ○ Requires multiple primers ○ Produces discontinuous segments of complementary DNA → Okazaki fragments Termination DNA Polymerase I proofreads DNA molecule ○ Removes RNA primers via nuclease activity ○ Replaces with DNA nucleotides ○ Leaves nick or break along sugar-phosphate backbone Ligase seals the nicks Ends when all parent DNA nucleotides have been complemented ○ Protein complexes dissociate ○ Final products are two daughter molecules LG 2.3 Transcription and Post-Transcriptional Processes Topic Summary Transcription segment of DNA directs synthesis of RNA RNA polymerase unwinds DNA Complementary base pairing between incoming nucleotides and DNA template RNA strand does not remain hydrogen-bonded to DNA template RNA molecules are shorter than DNA molecules RNA and DNA Polymerase RNA Polymerase Catalyzes linkage of ribonucleotides Can start RNA chain without primer Less accurate DNA Polymerase Catalyzes linkage of deoxyribonucleotides Requires primer High accuracy Binding of transcription factors activated by promoters → unique regions in DNA ○ Makes binding of RNA polymerase possible Different Types of RNAs mRNA → messenger RNA, code for proteins rRNA → ribosomal; form basic ribosome structure; catalyzes protein synthesis tRNA → transfer adaptors between mRNA and amino acids snRNA → small nuclear; in different nuclear processes like mRNA splicing snoRNA → small nucleolar; process and modify rRNA Stages of Transcription Initiation I. Prokaryote ○ RNA Polymerase can recognize and bind to promoter ○ No initiation complex ○ Consensus sequence of the promoter (AT-rich regions): Pribnow box - TATAAT II. Eukaryote  ○ Transcription factors mediate RNA polymerase binding and initiation ○ TFs + RNAP II = Transcription Initiation Complex ○ Consensus sequence of promoter → TATA box - TATAAA Elongation Single gene can be transcribed simultaneously DNA is double stranded → sense strand (coding/non-template strand) and antisense strand (non-coding/template strand) RNA nucleotides complementary to template strand are added to 3’ end of strand DNA unwinds 10-20 bases for pairing with RNA nucleotides → double helix reforms Termination Transcription stops at terminator ○ In prokaryotes → proceeds through termination sequence in DNA ○ In eukaryotes → RNAP II continues well past termination signal Rho factor protein binds to termination site ○ Prokaryote Rho factor recognizes RNA rut (C-rich), upstream of the real terminator sequence Terminator serves as termination signal mRNA can be translated without further modification Rho catches up with RNAP and allows release ○ Eukaryote RNAP II continues along DNA strand to reach terminator sequence Polyadenylation signal sequence transcribes the polyadenylation signal (AAUAAA) pre-mRNA is released after 10-35 nucleotides downstream from AAUAAA RNAP II continues to transcribe and eventually dissociates Post-Transcriptional Processing in Eukaryotes RNAP II also bears pre-mRNA-processing proteins on its tails  ○ Transferred to nascent RNA Production and Alteration of Production of mRNA mRNA Introns (non-coding sequences) in between exons (coding sequences) Introns loop out as snRNP (small nuclear ribonucleoprotein particles ○ Binds to signals at the end of each intron Joins with other proteins to form spliceosome Introns are removed and exons are spliced together Alteration of mRNA 5’ capping 3’ polyadenylation or addition of poly-A tail UTRs (untranslated regions) for ribosome binding signals Functions ○ Export of mRNA from nucleus to cytoplasm ○ Prevent degradation of mRNA by hydrolytic enzymes ○ Signals rRNA attachment at 5’ end LG 2.4 Translation and Post-Translational Processes Topic Summary The Genetic Code Gene-to-protein information flow based on triplet code / codon → sequence of non-overlapping, three-nucleotide bases ○ Each base is only part of one codon ○ Transcribed into complementary mRNA → translated into amino acid chain; forming polypeptide Continuous Requires instructions for starting or stopping translation ○ AUG → start codon ○ UAA, UAG, UGA → stop codons 64 triplets Universal  Redundant, but not ambiguous ○ No two amino acids are coded by the same codon Wobble effect → degenerate codons coding for same amino acid differ only by the base in the third nucleotide Codons must be read in correct reading frame for specific polypeptide to be formed Overview of Translation Synthesis of polypeptide using genetic info in mRNA ○ Ribonucleotides to amino acids Requires mRNA, tRNA, rRNA, enzymes and protein factors, energy (ATP or GTP) tRNA 70-80 nucleotides long Cloverleaf characteristic structures Fold into compact L shapes → for them to fit in ribosomes during translation Interprets mRNA by acting as adaptor molecule → recognizes codon and encoded amino acids Transfers amino acids to rRNA Anticodon loop at end of folded tRNA base → paired with complementary codon on mRNA template Ribosome Complex where protein synthesis occurs Consists of large and small subunit → composed of ribosomal proteins and rRNA Facilitates coupling of tRNA anticodon with mRNA codon Has 3 binding sites for tRNA ○ A site (aminoacyl-tRNA) → holds tRNA carrying next amino acid to be added ○ P site (peptidyl-tRNA) → holds tRNA carrying growing polypeptide chain ○ E site → exit site for discharged tRNA Stages of Translation Initiation Direction is 5’ to 3’ ○ Prokaryote mRNA is polycistronic → single mRNA molecule may code for more than one peptide  Ribosomal Binding Site: Shine-Dalgarno Sequence → present in 5’ UTRs: AGGAGG ○ Eukaryote Monocistronic → each mRNA codes for only one peptide Kozak Consensus Sequence → bases around initiating AUG influence the efficiency: GCCRCCAUGG Brings together mRNA, tRNA with first amino acid and two ribosomal subunits ○ mRNA binds to small ribosomal subunit of rRNA (ribosomal binding site) ○ Initiator tRNA anticodon (UAC) carrying amino acid methionine binds to start codon (AUG) ○ Large ribosomal subunit of rRNA binds to complete translation initiation complex Elongation N-terminal to C-terminal direction Amino acids added to C-terminus of growing polypeptide chain Each addition includes proteins called elongation factors ○ Codon recognition → incoming aminoacyl tRNA binds to the codon in the A site ○ Peptide bond formation → Peptidyl-transferase joins polypeptide from P site to newly arrived amino acid in A site ○ Translocation → tRNA in A site with growing polypeptide is translocated to P site; tRNA moves to E site Termination When stop codon (UAA, UAG, UGA) of mRNA reaches A site of ribosome ○ A site recognizes protein called release factor instead of tRNA ○ Hydrolyzes bond between tRNA in P site and last amino acid of polypeptide chain Last tRNA, mRNA, and polypeptide chain released ○ Two ribosomal subunits separated ○ Assembly by other protein factors dissociate Post-Translational Processing mRNA is translated simultaneously by multiple ribosomes (polyribosomes) ○ Both prokaryotic and eukaryotic cells Attachment of carbohydrates, lipids, phosphate groups and others are possible now Enzymes may remove one or more amino acids from N-terminus of polypeptide chain Single chain may also be enzymatically cleaved ○ Can also bind together to become subunits of quaternary-structure protein Protein folding is performed simultaneously based on amino acid sequence ○ Aided by chaperonins or chaperone proteins ○ Does not specify the final protein structure Ribosomes are interchangeable → can be bound or free Signal recognition particle → part off complex that identifies signal peptide and carries ribosome to endoplasmic reticulum through multi protein translocation LG 2.6 Control of Gene Expression Topic Summary Regulation of Gene Central Dogma demonstrates transfer of genetic information; Expression in Prokaryotes gene expression must also be regulated Amounts of different proteins instead of the production of different types Related genes in prokaryotes clustered into mRNA by RNA polymerase ○ Promoter is at the end of cluster Negative Inducible System Negative transcriptional control system → repressor proteins produced by regulatory gene Lactose operon (lac operon) Operator Operon → relation between operator gene and structural gene Lac repressor binds to operator Prevents transcription When allolactose (inducer) (rearranged lactose) is present, it  makes repressor let go of operator Promoter Attachment of RNAP to promoter is controlled by second side CRP ○ cAMP receptor protein or CAP, catabolite activator protein ○ Positive regulator → enhances function Attachment of CRP relies on presence of cAMP Without cAMP, CAP does not bind and makes the transcription occur at a low rate Negative Repressible System Positive transcriptional control system → expressor protein Tryptophan operon (trp operon) ○ Controlled by trp regulatory gene (trp R) ○ When tryptophan (co-repressor)is present, repressor binds to operator and synthesis is blocked ○ No tryptophan → Repressor dissociates ○ RNAP is between operator and structural genes Operator region is not transcribed into RNA LG 2.7 Control of Gene Expressions (Mutations) Topic Summary Mutations Change in protein during gene expression caused by alteration of DNA Production of new genes, source of genetic diversity How are Mutations Formed? Mutagenesis  Mishaps in DNA replication, spontaneous or induced Mutagens → induce mutation Ionizing radiation (Uv rays, nuclear fallout radiation) Viruses, microorganisms Alcohol and dietary components Environmental poisons and irritants Also carcinogens Inheritance of Mutation Not all mutations are inherited Gametic mutations only Somatic mutations → form chimera, individual with genetically varied cells Beneficial, Harmful, Neutral Hemoglobin beta mutation → sickle cell anemia; harmful Mutations Tolerance for high levels of blood cholesterol → alteration of amino acid in functioning protein; beneficial neutral/silent → hard to detect; no new protein products, no change in phenotype ○ Due to degeneracy of genetic code → having several codons code for same amino acid Gene Mutations Localized changes in DNA strand; nucleotide level Point mutation → change in one nucleotide or Change to triplet nucleotides Altered DNA, mRNA, protein chain May be due to complementary base pair mismatching 1. Missense substitution → New amino acid produced 2. Nonsense substitution → codon may code for amino acid that signals transcription to stop Reading Frameshifts Nucleotides are displaced → new set of codons in sequence May be caused by insertion or deletion of nucleotide base Partial reading frameshift → resolution in both insertion and deletion Chromosome Mutations Block mutations; whole blocks of genes during meiosis Changes in sequence and number of sets of genes in chromosomes 1. Deletion → break in chromosome at two points; shorter chromosome  ○ Midsection falls 2. Inversion → midsection rotates 180 degrees and rejoins chromosome 3. Insertion → gene section joins another chromosome 4. Duplication → section rejoins homologue of original chromosome 5. Translocation → groups of genes fall and join another chromosome LG 2.8 Exceptions to the Central Dogma (Part 1) Topic Summary Features of Virion Virion → complete virus particle Capsomeres that make up capsid ○ Capsid → protein coat enclosing genomic material (DNA or DNA) More sophisticated virions have membranous envelope and other appendages Acellular Entities Allow them to obligate intracellular parasites Need hosts to thrive Host-specific → limited number of species and tissue types for host range Phages Bacterial viruses Used in most studies, bacterial processes are easier to work on Viral infection and viral replicative cycles Viral Infection 1. Attachment of virion to host cell 2. Penetration of host cell membrane or release nucleic acid in host’s cytoplasm 3. Uncoating of virion’s capsid to reveal genomic material 4. Replication of genomic material, transcription and translation of viral proteins 5. Assembly of viral particles forming progeny virions 6. Release of progeny virions Viral Replicative Cycle Lysogenic Cycle  Aka temperate cycle Virus does not immediately kill infected cell → allows its genetic material to be incorporated into host’s genome Viral genome replicates while cell is alive → viral infection is latent/hidden Until activated into lytic cycle Lytic Cycle Virus causes infection → virulent upon release of virion progeny Host cell lysed (broken apart) for progeny to go infect others LG 2.9 Exceptions to the Central Dogma (Part 2) Topic Summary Classification Based on Dictate replication cycle → steps in central dogma will vary in Genomic Material order, may be omitted, or other mechanisms added Classification 1. Class/Family I → double-stranded DNA 2. Class II → single-stranded DNA 3. Class III → double-stranded RNA 4. Class IV → single-stranded positive sense RNA serving as mRNA 5. Class V → single-stranded RNA as template for mRNA synthesis 6. Class VI → single-stranded RNA used for DNA synthesis SARS-CoV-2 Replication Type IV virus Cycle Doesn’t need DNA, only production of RNA needed Viral RNA hijacks host’s ribosomes → allows translation and production of necessary proteins for virus; building capsid and viral membranes Single-stranded negative sense RNA → uses RNA to make mRNA Single-stranded positive sense RNA → RNA as mRNA Retrovirus Replication Cycle Type VI viruses Reverse transcription → reversing DNA replication and transcription of RNA ○ Use single-stranded RNA genomes and reverse transcriptase enzymes Discovered in 1970 by Howard Temin and David Baltimore ○ Central dogma used to be the absolute truth ○ Discovery impacted studies on AIDS caused by HIV, a retrovirus Reverse Transcription Allows SARS-CoV-2 identification Implication ○ PCR tests are Real-time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) ○ SARS-CoV-2 has no DNA, but our genomic technology only allows production of numerous DNA copies ○ Viral genomic samples need to be taken as templates to create corresponding DNA templates Other Entities Do not follow central dogma Viroids → plant pathogens, virus-like Single-stranded circular RNA particles only able to replicate within host cells Virusoids → like viroids but do note code for any proteins that need helper viruses to replicate in host cells Prions → misfolded protein entities No DNA or RNA Able to cause infections causing fatal neurodegenerative diseases