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Nucleic Acids: Cell Overview and Core Topics

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Outline I.Cellular Overview II.Anatomy of the Nucleic Acids 1.Building blocks 2.Structure (DNA, RNA) III.Looking at the Central Dogma 1.DNA Replication 2.RNA Transcription 3.Protein Synthesis

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Cellular Overview DNA and RNA in the Cell

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Classes of Nucleic Acids: DNA DNA is usually found in the nucleus Small amounts are also found in: mitochondria of eukaryotes chloroplasts of plants Packing of DNA: 2-3 meters long histones genome = complete collection of hereditary information of an organism

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Classes of Nucleic Acids: RNA FOUR TYPES OF RNA mRNA - Messenger RNA tRNA - Transfer RNA rRNA - Ribosomal RNA snRNA - Small nuclear RNA

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Anatomy of Nucleic Acids THE BUILDING BLOCKS

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Nucleic acids are linear polymers. Each monomer consists of: 1. a sugar 2. a phosphate 3. a nitrogenous base

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Nitrogenous Bases

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DNA (deoxyribonucleic acid): adenine (A)guanine (G) cytosine (C)thymine (T) RNA (ribonucleic acid): adenine (A)guanine (G) cytosine (C)uracil (U) Why ?

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Properties of purines and pyrimidines: 1.keto enol tautomerism 2.strong UV absorbance

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Pentoses of Nucleic Acids This difference in structure affects secondary structure and stability. Which is more stable?

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Nucleosides linkage of a base and a sugar.

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Nucleotides - nucleoside + phosphate - monomers of nucleic acids - NA are formed by 3-to-5 phosphodiester linkages

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Shorthand notation: - sequence is read from 5 to 3 - corresponds to the N to C terminal of proteins

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Nucleic Acids: Structure DNA

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Primary Structure nucleotide sequences

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DNA Double Helix Maurice Wilkins and Rosalind Franklin James Watson and Francis Crick Features: two helical polynucleotides coiled around an axis chains run in opposite directions sugar-phosphate backbone on the outside, bases on the inside bases nearly perpendicular to the axis repeats every 34 10 bases per turn of the helix diameter of the helix is 20 Secondary Structure

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Double helix stabilized by hydrogen bonds. Which is more stable?

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Axial view of DNA

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A and B forms are both right-handed double helix. A-DNA has different characteristics from the more common B-DNA.

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left-handed backbone phosphates zigzag Z-DNA

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Comparison Between A, B, and Z DNA: A-DNA: right-handed, short and broad, 11 bp per turn B-DNA: right-handed, longer, thinner, 10 bp per turn Z-DNA: left-handed, longest, thinnest, 12 bp per turn

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Major and minor grooves are lined with sequence-specific H-bonding.

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Supercoiling relaxed DNA supercoiled DNA Tertiary Structure

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Topoisomerase I relaxation of supercoiled structures

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Topoisomerase II add negative supercoils to DNA

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Consequences of double helical structure: 1. Facilitates accurate hereditary information transmission 2.Reversible melting melting: dissociation of the double helix melting temperature (T m ) hypochromism annealing

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Structure of Single-stranded DNA Stem Loop

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Nucleic Acids: Structure RNA

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Secondary Structure transfer RNA (tRNA) : Brings amino acids to ribosomes during translation

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Transfer RNA Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem Only one tRNA structure (alone) is known Many non-canonical base pairs found in tRNA

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ribosomal RNA (rRNA) : Makes up the ribosomes, together with ribosomal proteins. Ribosomes synthesize proteins All ribosomes contain large and small subunits rRNA molecules make up about 2/3 of ribosome Secondary structure features seem to be conserved, whereas sequence is not There must be common designs and functions that must be conserved

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messenger RNA (mRNA) : Encodes amino acid sequence of a polypeptide

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small nuclear RNA (snRNA) :With proteins, forms complexes that are used in RNA processing in eukaryotes. (Not found in prokaryotes.)

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Central Dogma DNA Replication, Recombination, and Repair

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Central Dogma

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DNA Replication process of producing identical copies of original DNA strand separation followed by copying of each strand fixed by base-pairing rules

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DNA replication is semi-conservative.

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DNA replication is bidirectional. involves two replication forks that move in opposite direction

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DNA Replication Begins at specific start sites in E. coli, origin of replication, oriC locus binding site for dnaA, initiation protein rich in A-T

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DNA replication requires unwinding of the DNA helix. expose single-stranded templates DNA gyrase acts to overcome torsional stress imposed upon unwinding helicases catalyze unwinding of double helix -disrupts H-bonding of the two strands SSB (single-stranded DNA-binding proteins) binds to the unwound strands, preventing re-annealing

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Primer RNA primes the synthesis of DNA. Primase synthesizes short RNA.

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DNA replication is semidiscontinuous DNA polymerase synthesizes the new DNA strand only in a 5 3 direction. Dilemma: how is 5 3 copied? The leading strand copies continuously The lagging strand copies in segments called Okazaki fragments (about 1000 nucleotides at a time) which will then be joined by DNA ligase

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Overall: each of the two DNA duplexes contain one old and one new DNA strand (semi-conservative) and half of the new strand was formed by leading strand and the other half by lagging strand.

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DNA Polymerase = enzymes that replicate DNA All DNA Polymerases share the following: 1.Incoming base selected in the active site (base-complementarity) 2.Chain growth 5 3 direction (antiparallel to template) 3.Cannot initiate DNA synthesis de novo (requires primer) First DNA Polymerase discovered E.coli DNA Polymerase I (by Arthur Kornberg and colleagues) Roger D. Kornberg 2006 Nobel Prize in Chemistry Arthur Kornberg 1959 Nobel Prize in Physiology and Medicine http://www.nobelprize.org

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DNA Polymerase specificity dictated by H-bonding and shape complementarity between bases binding of correct base is favorable (more stable) interaction of residues in the enzyme to the minor groove of DNA close down around the incoming NTP

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Mechanism of DNA linkage:

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3 5 exonuclease activity - removes incorrect nucleotides from the 3-end of the growing chain (proofreader and editor) - polymerase cannot elongate an improperly base-paired terminus proofreading mechanisms Klenow fragment removes mismatched nucleotides from the 3 end of DNA (exonuclease activity) detection of incorrect base - incorrect pairing with the template (weak H-bonding) - unable to interact with the minor groove (enzyme stalls)

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5 3 exonuclease activity Exonuclease activity - remove distorted segments lying in the path of the advancing polymerase

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DNA Ligase = seals the nicks between Okazaki fragments DNA ligase seals breaks in the double stranded DNA DNA ligases use an energy source (ATP in eukaryotes and archaea, NAD + in bacteria) to form a phosphodiester bond between the 3 hydroxyl group at the end of one DNA chain and 5-phosphate group at the end of the other.

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DNA replication terminates at the Ter region. the oppositely moving replication forks meet here and replication is terminated contain core elements 5-GTGTGTTGT binds termination protein (Tus protein)

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Eukaryotic DNA Replication Like E. coli, but more complex Human cell: 6 billion base pairs of DNA to copy Multiple origins of replication: 1 per 3000-30000 base pairs E.coli 1 chromosome Human 23 E.coli circular chromosome; Human linear

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Telomeres The Ends of Linear DNA Possess Telomeres Present because DNA is shortened after each round of replication Contains hundreds of tandem repeats of a hexanucleotide sequence (AGGGTT in humans) Telomeres at the 3 end is G rich and is slightly longer May form large loops to protect chromosome ends

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DNA Recombination = recombinases Holliday junction crosslike structure natural process of genetic rearrangement

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Mutations 1. Substitution of base pair a.transition b.transversion 2. Deletion of base pair/s 3. Insertion/Addition of base pair/s DNA replication error rate: 3 bp during copying of 6 billion bp Macrolesions: Mutations involving changes in large portions of the genome

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Agents of Mutations 1. Physical Agents a)UV Light b) Ionizing Radiation 2. Chemical Agents Some chemical agents can be classified further into a)Alkylating b)Intercalating c)Deaminating 3. Viral

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UV Light Causes Pyrimidine Dimerization Replication and gene expression are blocked

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Chemical mutagens 5-bromouracil and 2-aminopurine can be incorporated into DNA

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Deaminating agents Ex: Nitrous acid (HNO 2 ) Converts adenine to hypoxanthine, cytosine to uracil, and guanine to xanthine Causes A-T to G-C transitions

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Alkylating agents

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Intercalating agents

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Acridines Intercalate in DNA, leading to insertion or deletion The reading frame during translation is changed

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DNA Repair Direct repair Photolyase cleave pyrimidine dimers Base excision repair E. coli enzyme AlkA removes modified bases such as 3- methyladenine (glycosylase activity is present) Nucleotide excision repair Excision of pyrimidine dimers (need different enzymes for detection, excision, and repair synthesis)

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Do we has a quiz?

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QUIZ 1.Draw the structure of any nitrogenous base of your picking. (1 pt) 2. What is the difference between the glycosidic bond and the phosphodiester bond? (2 pts) 3.Give the reason why DNA utilizes the deoxyribose while RNA uses the ribose. (2 pts) 4.Enumerate all the enzymes and proteins involved in DNA replication and briefly state their importance/function. A short concise answer will suffice. (4 pts) 5.Give the partner strand of this piece of DNA: 5-ACTCATGATTAGCAG-3 (1 pt)

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Central Dogma RNA Transcription

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Process of Transcription has four stages: 1.Binding of RNA polymerase at promoter sites 2.Initiation of polymerization 3.Chain elongation 4.Chain termination

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Transcription (RNA Synthesis) RNA Polymerases Template (DNA) Activated precursors (NTP) Divalent metal ion (Mg 2+ or Mn 2+ ) Mechanism is similar to DNA Synthesis

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Reece R. Analysis of Genes and Genomes.2004. p47. Limitations of RNAP II: 1.It cant recognize its target promoter and gene. (BLIND) 2.It is unable to regulate mRNA production in response to developmental and environmental signals. (INSENSITIVE)

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Start of Transcription Promoter Sites Where RNA Polymerase can indirectly bind

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TATA box a DNA sequence (5TATAA3) found in the promoter region of most eukaryotic genes. Abeles F, et al. Biochemistry. 1992. p391. Preinitiation Complex (PIC) Transcription Factors (TF): Hampsey M. Molecular Genetics of RNAP. Microbiology and Molecular Biology Reviews. 1998. p7. TFIIDbinds to TATA; promotes TFIIB binding TFIIAstabilizes TBP binding TFIIBpromotes TFIIF-pol II binding TFIIFtargets pol II to promoter TFIIEstimulates TFIIH kinase and ATPase actiivities TFII Hhelicase, ATPase, CTD kinase activities

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Termination of Transcription Terminator Sequence Encodes the termination signal In E. coli base paired hair pin (rich in G C) followed by UUU 1. Intrinsic termination = termination sites causes the RNAP to pause causes the RNA strand to detach from the DNA template

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Termination of Transcription 2. Rho termination = Rho protein,

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prokaryotes: transcription and translation happen in cytoplasm eukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)

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In eukaryotes, mRNA is modified after transcription Capping, methylation Poly-(A) tail, splicing capping: guanylyl residue capping and methylation ensure stability of the mRNA template; resistance to exonuclease activity

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Eukaryotic genes are split genes: coding regions (exons) and noncoding regions (introns)

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Introns & Exons Introns Intervening sequences Exons Expressed sequences

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Splicing Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs) splicing occurs in the spliceosome!

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Reverse Transcription RNA-Directed DNA Polymerase 1964: Howard Temin notices that DNA synthesis inhibitors prevent infection of cells in culture by RNA tumor viruses. Temin predicts that DNA is an intermediate in RNA tumor virus replication 1970: Temin and David Baltimore (separately) discover the RNA-directed DNA polymerase - aka "reverse transcriptase"

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Reverse Transcriptase Primer required, but a strange one - a tRNA molecule that the virus captures from the host RT transcribes the RNA template into a complementary DNA (cDNA) to form a DNA:RNA hybrid All RNA tumor viruses contain a reverse transcriptase

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RT II Three enzyme activities RNA-directed DNA polymerase RNase H activity - degrades RNA in the DNA:RNA hybrids DNA-directed DNA polymerase - which makes a DNA duplex after RNase H activity destroys the viral genome HIV RT: very error-prone (1 bp /2000 to 4000 bp) HIV therapy: AZT (or 3'-azido-2',3'- dideoxythymidine) specifically inhibits RT

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Central Dogma Translation: Protein Synthesis

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Translation Starring three types of RNA 1.mRNA 2.tRNA 3.rRNA

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Properties of mRNA 1.In translation, mRNA is read in groups of bases called codons 2.One codon is made up of 3 nucleotides from 5 to 3 of mRNA 3.There are 64 possible codons 4.Each codon stands for a specific amino acid, corresponding to the genetic code 5.However, one amino acid has many possible codons. This property is termed degeneracy 6.3 of the 64 codons are terminator codons, which signal the end of translation

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Genetic Code 3 nucleotides (codon) encode an amino acid The code is nonoverlapping The code has no punctuation

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Synonyms Different codons, same amino acid Most differ by the last base XYC & XYU XYG & XYA Minimizes the deleterious effect of mutation

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Encoded sequences. (a) Write the sequence of the mRNA molecule synthesized from a DNA template strand having the sequence (b) What amino acid sequence is encoded by the following base sequence of an mRNA molecule? Assume that the reading frame starts at the 5 end. Practice

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Answers (a) 5 -UAACGGUACGAU-3. (b) Leu-Pro-Ser-Asp-Trp-Met.

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tRNA as Adaptor Molecules Amino acid attachment site Template recognition site Anticodon Recognizes codon in mRNA

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tRNA as Adaptor Molecules

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Mechanics of Protein Synthesis All protein synthesis involves three phases: initiation, elongation, termination Initiation involves binding of mRNA and initiator aminoacyl-tRNA to small subunit(30S), followed by binding of large subunit (50S) of the ribosome Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites. Termination occurs when "stop codon" reached

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Translation Occurs in the ribosome Prokaryote START fMet (formylmethionine) bound to initiator tRNA Recognizes AUG and sometimes GUG (but they also code for Met and Val respectively) AUG (or GUG) only part of the initiation signal; preceded by a purine-rich sequence

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Translation Eukaryote START AUG nearest the 5 end is usually the start signal

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Termination Stop signals (UAA, UGA, UAG): recognized by release factors (RFs) hydrolysis of ester bond between polypeptide and tRNA

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Reference: Garrett, R. and C. Grisham. Biochemistry. 3 rd edition. 2005. Berg, JM, Tymoczko, JL and L. Stryer. Biochemistry. 5 th edition. 2002.