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Nucleic Acids: Cell Overview and Core Topics. Outline Cellular Overview Anatomy of the Nucleic Acids Building blocks Structure (DNA, RNA ) Looking at the Central Dogma DNA Replication RNA Transcription Protein Synthesis. DNA and RNA in the Cell. Cellular Overview.

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Presentation Transcript

  • Outline

  • Cellular Overview

  • Anatomy of the Nucleic Acids

    • Building blocks

    • Structure (DNA, RNA)

  • Looking at the Central Dogma

    • DNA Replication

    • RNA Transcription

    • Protein Synthesis


Cellular overview

DNA and RNA in the Cell

Cellular Overview


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


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 acids

THE BUILDING BLOCKS

Anatomy of Nucleic Acids


Nucleic acids are linear polymers.

Each monomer consists of:

1. a sugar

2. a phosphate

3. a nitrogenous base



Nitrogenous Bases

DNA (deoxyribonucleic acid):

adenine (A) guanine (G)

cytosine (C) thymine (T)

Why ?

RNA (ribonucleic acid):

adenine (A) guanine (G)

cytosine (C) uracil (U)



Pentoses of Nucleic Acids

This difference in structure affects secondary structure and stability.

Which is more stable?


Nucleosides

linkage 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

DNA

Nucleic Acids: Structure


Primary Structure

  • nucleotide sequences


Secondary Structure

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 Å

  • 10 bases per turn of the helix

  • diameter of the helix is 20 Å




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

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


Z-DNA

  • left-handed

  • backbone phosphates zigzag


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



  • Consequences of double helical structure: H-bonding.

  • 1. Facilitates accurate hereditary information transmission

  • Reversible melting

    • melting: dissociation of the double helix

    • melting temperature (Tm)

    • hypochromism

    • annealing


Tertiary Structure H-bonding.

Supercoiling

supercoiledDNA

relaxed DNA



Topoisomerase H-bonding.II – add negative supercoils to DNA


Structure of Single-stranded DNA H-bonding.

Stem Loop


Nucleic acids structure1

RNA H-bonding.

Nucleic Acids: Structure


Secondary Structure H-bonding.

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


  • Transfer RNA H-bonding.

  • 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 ( H-bonding.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


messenger RNA (mRNA) H-bonding.: Encodes amino acid sequence of a polypeptide


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


Central dogma

DNA Replication, H-bonding.Transcription, and Translation

Central Dogma


Central Dogma H-bonding.


Dna replication

Central Dogma H-bonding.

DNA Replication




DNA replication is bidirectional. original DNA

  • involves two replication forks that move in opposite direction


DNA Replication original DNA

  • Begins at specific start sites

    • in E. coli, origin of replication, oriC locus

    • binding site for dnaA, initiation protein

    • rich in A-T


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 strandand the other half by lagging strand.


  • DNA replication requires unwinding of the DNA helix. and one “new” DNA strand

    • 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


Primer and one “new” DNA strand

RNA primes the synthesis of DNA.

Primase synthesizes short RNA.


  • DNA replication is and one “new” DNA strand 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


DNA Polymerase and one “new” DNA strand

= enzymes that replicate DNA

  • All 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)

Arthur Kornberg

1959 Nobel Prize in Physiology and Medicine

Roger D. Kornberg

2006 Nobel Prize in Chemistry

http://www.nobelprize.org


DNA Polymerase and one “new” DNA strand

  • 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: and one “new” DNA strand


3 and one “new” DNA strand ’  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)


Exonuclease and one “new” DNA strand activity

5’  3’ exonuclease activity

  • remove distorted segments lying in the path of the advancing polymerase


DNA Ligase and one “new” DNA strand

= 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.


  • DNA replication terminates at the and one “new” DNA strand 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 and one “new” DNA strand

  • 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


Telomeres and one “new” DNA strand

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


DNA Recombination = and one “new” DNA strand

natural process of genetic rearrangement

  • recombinases

  • Holliday junction – crosslike structure


  • Mutations and one “new” DNA strand

  • Substitution of base pair

    • transition

    • transversion

  • Deletion of base pair/s

  • Insertion/Addition of base pair/s

Macrolesions: Mutations involving changes in large portions of the genome

DNA replication error rate: 3 bp during copying of 6 billion bp


  • Agents of Mutations and one “new” DNA strand

  • Physical Agents

    • UV Light

    • Ionizing Radiation

  • Chemical Agents

    • Some chemical agents can be classified further into

    • Alkylating

    • Intercalating

    • Deaminating

  • Viral


UV Light Causes Pyrimidine Dimerization and one “new” DNA strand

  • Replication and gene expression are blocked


  • Chemical and one “new” DNA strand mutagens

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


  • Deaminating and one “new” DNA strand agents

    • Ex: Nitrous acid (HNO2)

    • Converts adenine to hypoxanthine, cytosine to uracil, and guanine to xanthine

    • Causes A-T to G-C transitions




  • Acridines and one “new” DNA strand

    • Intercalate in DNA, leading to insertion or deletion

    • The reading frame during translation is changed


DNA Repair and one “new” DNA strand

  • 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)


Do we has a quiz? and one “new” DNA strand


QUIZ and one “new” DNA strand

  • Draw 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)


Rna transcription

Central Dogma and one “new” DNA strand

RNA Transcription


Process of Transcription has and one “new” DNA strand four stages:

  • Binding of RNA polymerase at promoter sites

  • Initiation of polymerization

  • Chain elongation

  • Chain termination


Transcription (RNA Synthesis) and one “new” DNA strand

  • RNA Polymerases

    • Template (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. and one “new” DNA strand

  • Limitations of RNAP II:

  • It can’t recognize its target promoter and gene. (BLIND)

  • It is unable to regulate mRNA production in response to developmental and environmental signals. (INSENSITIVE)


Start of Transcription and one “new” DNA strand

  • Promoter Sites

    • Where RNA Polymerase can indirectly bind


Preinitiation Complex (PIC) and one “new” DNA strand

TATA box – a DNA sequence (5’—TATAA—3’) found in the promoter region of most eukaryotic genes.

Abeles F, et al. Biochemistry. 1992. p391.

Transcription Factors (TF):

Hampsey M. Molecular Genetics of RNAP. Microbiology and Molecular Biology Reviews. 1998. p7.


Termination of Transcription and one “new” DNA strand

1. Intrinsic termination = termination sites

  • Terminator Sequence

    • Encodes the termination signal

    • In E. coli – base paired hair pin (rich in GC) followed by UUU…

causes the RNAP to pause

causes the RNA strand to detach from the DNA template


Termination of Transcription and one “new” DNA strand

2. Rho termination = Rho protein, ρ


prokaryotes and one “new” DNA strand : transcription and translation happen in cytoplasm

eukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)


capping: guanylyl residue

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


Eukaryotic genes are split genes: coding regions and one “new” DNA strand (exons) and noncoding regions (introns)


Introns and one “new” DNA strand & Exons

  • Introns

    • Intervening sequences

  • Exons

    • Expressed sequences


Splicing and one “new” DNA strand

Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs)

splicing occurs in the spliceosome!


Reverse Transcription and one “new” DNA strand

  • 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"


Reverse Transcriptase and one “new” DNA strand

  • 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 and one “new” DNA strand

  • 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


Translation protein synthesis

Central Dogma and one “new” DNA strand

Translation: Protein Synthesis


Translation and one “new” DNA strand

Starring three types of RNA

  • mRNA

  • tRNA

  • rRNA


Properties of mRNA and one “new” DNA strand

  • In 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 Code and one “new” DNA strand

  • 3 nucleotides (codon) encode an amino acid

  • The code is nonoverlapping

  • The code has no punctuation


Synonyms and one “new” DNA strand

  • Different codons, same amino acid

  • Most differ by the last base

    • XYC & XYU

    • XYG & XYA

  • Minimizes the deleterious effect of mutation


Practice and one “new” DNA strand

  • 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.


Answers and one “new” DNA strand

  • (a) 5’ -UAACGGUACGAU-3’ .

  • (b) Leu-Pro-Ser-Asp-Trp-Met.


tRNA and one “new” DNA strand as Adaptor Molecules

  • Amino acid attachment site

  • Template recognition site

    • Anticodon

      • Recognizes codon in mRNA


tRNA and one “new” DNA strand as Adaptor Molecules


Mechanics of Protein Synthesis and one “new” DNA strand

  • 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.

  • Terminationoccurs when "stop codon" reached


Translation and one “new” DNA strand

  • 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


Translation and one “new” DNA strand

  • Eukaryote START

    • AUG nearest the 5’ end is usually the start signal


Termination and one “new” DNA strand

  • Stop signals (UAA, UGA, UAG):

    • recognized by release factors (RFs)

    • hydrolysis of ester bond between polypeptide and tRNA


Reference: and one “new” DNA strand

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

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


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