Nucleic Acids: Cell Overview and Core Topics

Nucleic Acids: Cell Overview and Core TopicsOutline

Cellular Overview

Anatomy of the Nucleic AcidsBuilding blocksStructure (DNA, RNA)

Looking at the Central DogmaDNA ReplicationRNA TranscriptionProtein Synthesis

2Cellular OverviewDNA and RNA in the CellClasses 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

Classes of Nucleic Acids: RNA FOUR TYPES OF RNA

mRNA - Messenger RNA

tRNA - Transfer RNA

rRNA - Ribosomal RNA

snRNA - Small nuclear RNA

Anatomy of Nucleic AcidsTHE BUILDING BLOCKS

Nucleic acids are linear polymers.Each monomer consists of:1. a sugar2. a phosphate3. a nitrogenous baseNitrogenous Bases

Nitrogenous BasesDNA (deoxyribonucleic acid):adenine (A)guanine (G)cytosine (C)thymine (T)RNA (ribonucleic acid):adenine (A)guanine (G)cytosine (C)uracil (U)Why ?Properties of purines and pyrimidines:

keto enol tautomerismstrong UV absorbance

Pentoses of Nucleic Acids

This difference in structure affects secondary structure and stability.Which is more stable?

Nucleosideslinkage of a base and a sugar.Nucleotides- nucleoside + phosphate

- monomers of nucleic acids - NA are formed by 3-to-5 phosphodiester linkages

Shorthand notation: sequence is read from 5 to 3 corresponds to the N to C terminal of proteins Nucleic Acids: Structure DNAPrimary Structure nucleotide sequences

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

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

Axial view of DNA

A and B forms are both right-handed double helix.

A-DNA has different characteristics from the more common B-DNA.

left-handed backbone phosphates zigzagZ-DNAComparison 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

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

Supercoilingrelaxed DNA supercoiled DNA Tertiary Structure

Topoisomerase I relaxation of supercoiled structures

Topoisomerase II add negative supercoils to DNAConsequences of double helical structure:

1. Facilitates accurate hereditary information transmission

Reversible melting melting: dissociation of the double helix melting temperature (Tm) hypochromism annealing

Structure of Single-stranded DNAStem LoopNucleic Acids: Structure RNASecondary Structuretransfer RNA (tRNA) : Brings amino acids to ribosomes during translation

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

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

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

Central DogmaDNA Replication, Recombination, and RepairCentral Dogma

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

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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55DNA 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 helixdisrupts H-bonding of the two strands

SSB (single-stranded DNA-binding proteins) binds to the unwound strands, preventing re-annealing

Primer

RNA primes the synthesis of DNA.

Primase synthesizes short RNA.

DNA replication is semidiscontinuous

DNA polymerase synthesizes the new DNA strand only in a 53 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.DNA Polymerase= enzymes that replicate DNAAll DNA Polymerases share the following:

Incoming base selected in the active site (base-complementarity)Chain growth 5 3 direction (antiparallel to template)Cannot initiate DNA synthesis de novo (requires primer)First DNA Polymerase discovered E.coli DNA Polymerase I (by Arthur Kornberg and colleagues)

Roger D. Kornberg2006 Nobel Prize in ChemistryArthur Kornberg1959 Nobel Prize in Physiology and Medicinehttp://www.nobelprize.org71DNA 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

Mechanism of DNA linkage: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)

5 3 exonuclease activity

Exonuclease activity

remove distorted segments lying in the path of the advancing polymeraseDNA Ligase = seals the nicks between Okazaki fragmentsDNA ligase seals breaks in the double stranded DNADNA 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.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)

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 pairsE.coli 1 chromosomeHuman 23E.coli circular chromosome; Human linear

Telomeres

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

DNA Recombination = recombinases Holliday junction crosslike structure

natural process of genetic rearrangementMutations

Substitution of base pairtransitiontransversion

Deletion of base pair/s

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 genomeAgents of Mutations

Physical AgentsUV Light Ionizing Radiation

Chemical AgentsSome chemical agents can be classified further intoAlkylatingIntercalatingDeaminating

Viral

UV Light Causes Pyrimidine DimerizationReplication and gene expression are blocked

85Chemical mutagens

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

Deaminating agentsEx: Nitrous acid (HNO2)Converts adenine to hypoxanthine, cytosine to uracil, and guanine to xanthineCauses A-T to G-C transitions

Alkylating agents

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Intercalating agents

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90AcridinesIntercalate in DNA, leading to insertion or deletionThe reading frame during translation is changed

DNA RepairDirect repairPhotolyase cleave pyrimidine dimersBase excision repairE. coli enzyme AlkA removes modified bases such as 3-methyladenine (glycosylase activity is present)Nucleotide excision repairExcision of pyrimidine dimers (need different enzymes for detection, excision, and repair synthesis)

Do we has a quiz?QUIZDraw the structure of any nitrogenous base of your picking. (1 pt)

What is the difference between the glycosidic bond and the phosphodiester bond? (2 pts)

Give the reason why DNA utilizes the deoxyribose while RNA uses the ribose. (2 pts)

Enumerate all the enzymes and proteins involved in DNA replication and briefly state their importance/function. A short concise answer will suffice. (4 pts)

Give the partner strand of this piece of DNA:5-ACTCATGATTAGCAG-3 (1 pt)Central DogmaRNA TranscriptionProcess of Transcription has four stages:Binding of RNA polymerase at promoter sitesInitiation of polymerizationChain elongationChain termination

Transcription (RNA Synthesis)RNA PolymerasesTemplate (DNA)Activated precursors (NTP)Divalent metal ion (Mg2+ or Mn2+)

Mechanism is similar to DNA Synthesis

Reece R. Analysis of Genes and Genomes.2004. p47.Limitations of RNAP II:It cant recognize its target promoter and gene. (BLIND)It is unable to regulate mRNA production in response to developmental and environmental signals. (INSENSITIVE)Start of TranscriptionPromoter SitesWhere RNA Polymerase can indirectly bind

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 bindingTFIIAstabilizes TBP bindingTFIIBpromotes TFIIF-pol II bindingTFIIFtargets pol II to promoterTFIIEstimulates TFIIH kinase and ATPase actiivitiesTFII Hhelicase, ATPase, CTD kinase activities

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Termination of TranscriptionTerminator SequenceEncodes the termination signalIn E. coli base paired hair pin (rich in GC) followed by UUU1. Intrinsic termination = termination sitescauses the RNAP to pausecauses the RNA strand to detach from the DNA template

Termination of Transcription2. Rho termination = Rho protein, prokaryotes: transcription and translation happen in cytoplasmeukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)

In eukaryotes, mRNA is modified after transcriptionCapping, methylationPoly-(A) tail, splicing

capping: guanylyl residuecapping and methylation ensure stability of the mRNA template; resistance to exonuclease activity

Eukaryotic genes are split genes: coding regions (exons) and noncoding regions (introns)Introns & ExonsIntronsIntervening sequencesExonsExpressed sequences

Splicing

Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs)splicing occurs in the spliceosome!Reverse TranscriptionRNA-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" 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 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 genomeHIV RT: very error-prone (1 bp /2000 to 4000 bp)HIV therapy: AZT (or 3'-azido-2',3'- dideoxythymidine) specifically inhibits RT Central DogmaTranslation: Protein SynthesisTranslationStarring three types of RNAmRNA

tRNA

rRNAProperties of mRNAIn translation, mRNA is read in groups of bases called codons

One codon is made up of 3 nucleotides from 5 to 3 of mRNA

There are 64 possible codons

Each codon stands for a specific amino acid, corresponding to the genetic code

However, one amino acid has many possible codons. This property is termed degeneracy

3 of the 64 codons are terminator codons, which signal the end of translation

Genetic Code3 nucleotides (codon) encode an amino acidThe code is nonoverlappingThe code has no punctuation

SynonymsDifferent codons, same amino acidMost differ by the last baseXYC & XYU XYG & XYAMinimizes the deleterious effect of mutationEncoded 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

Answers(a) 5 -UAACGGUACGAU-3 .(b) Leu-Pro-Ser-Asp-Trp-Met.tRNA as Adaptor MoleculesAmino acid attachment siteTemplate recognition siteAnticodonRecognizes codon in mRNA

tRNA as Adaptor Molecules

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133Mechanics 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 ribosomeElongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites. Termination occurs when "stop codon" reached TranslationOccurs in the ribosomeProkaryote STARTfMet (formylmethionine) bound to initiator tRNARecognizes 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

TranslationEukaryote START

AUG nearest the 5 end is usually the start signal

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

Garrett, R. and C. Grisham. Biochemistry. 3rd edition. 2005.

Berg, JM, Tymoczko, JL and L. Stryer. Biochemistry. 5th edition. 2002.