Nucleic acids cell overview and core topics
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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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Nucleic acids cell overview and core topics

Nucleic Acids: Cell Overview and Core Topics


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


Cellular overview

DNA and RNA in the Cell

Cellular Overview


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

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 cell overview and core topics

Nucleic acids are linear polymers.

Each monomer consists of:

1. a sugar

2. a phosphate

3. a nitrogenous base


Nucleic acids cell overview and core topics

Nitrogenous Bases


Nucleic acids cell overview and core topics

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)


Nucleic acids cell overview and core topics

  • Properties of purines and pyrimidines:

  • keto – enoltautomerism

  • strong UV absorbance


Nucleic acids cell overview and core topics

Pentoses of Nucleic Acids

This difference in structure affects secondary structure and stability.

Which is more stable?


Nucleic acids cell overview and core topics

Nucleosides

linkage of a base and a sugar.


Nucleic acids cell overview and core topics

Nucleotides

- nucleoside + phosphate

- monomers of nucleic acids

- NA are formed by 3’-to-5’ phosphodiester linkages


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

Primary Structure

  • nucleotide sequences


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

Double helix stabilized by hydrogen bonds.

Which is more stable?


Nucleic acids cell overview and core topics

Axial view of DNA


Nucleic acids cell overview and core topics

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

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


Nucleic acids cell overview and core topics

Z-DNA

  • left-handed

  • backbone phosphates zigzag


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

  • Consequences of double helical structure:

  • 1. Facilitates accurate hereditary information transmission

  • Reversible melting

    • melting: dissociation of the double helix

    • melting temperature (Tm)

    • hypochromism

    • annealing


Nucleic acids cell overview and core topics

Tertiary Structure

Supercoiling

supercoiledDNA

relaxed DNA


Nucleic acids cell overview and core topics

Topoisomerase I – relaxation of supercoiled structures


Nucleic acids cell overview and core topics

Topoisomerase II – add negative supercoils to DNA


Nucleic acids cell overview and core topics

Structure of Single-stranded DNA

Stem Loop


Nucleic acids structure1

RNA

Nucleic Acids: Structure


Nucleic acids cell overview and core topics

Secondary Structure

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


Nucleic acids cell overview and core topics

  • 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


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

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


Central dogma

DNA Replication, Transcription, and Translation

Central Dogma


Nucleic acids cell overview and core topics

Central Dogma


Dna replication

Central Dogma

DNA Replication


Nucleic acids cell overview and core topics

  • DNA Replication – process of producing identical copies of original DNA

    • strand separation followed by copying of each strand

    • fixed by base-pairing rules


Nucleic acids cell overview and core topics

DNA replication is semi-conservative.


Nucleic acids cell overview and core topics

DNA replication is bidirectional.

  • involves two replication forks that move in opposite direction


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

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.


Nucleic acids cell overview and core topics

  • 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


Nucleic acids cell overview and core topics

Primer

RNA primes the synthesis of DNA.

Primase synthesizes short RNA.


Nucleic acids cell overview and core topics

  • 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


Nucleic acids cell overview and core topics

DNA Polymerase

= 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


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

Mechanism of DNA linkage:


Nucleic acids cell overview and core topics

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)


Nucleic acids cell overview and core topics

Exonuclease activity

5’  3’ exonuclease activity

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


Nucleic acids cell overview and core topics

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.


Nucleic acids cell overview and core topics

  • 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)


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

DNA Recombination =

natural process of genetic rearrangement

  • recombinases

  • Holliday junction – crosslike structure


Nucleic acids cell overview and core topics

  • Mutations

  • 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


Nucleic acids cell overview and core topics

  • Agents of Mutations

  • Physical Agents

    • UV Light

    • Ionizing Radiation

  • Chemical Agents

    • Some chemical agents can be classified further into

    • Alkylating

    • Intercalating

    • Deaminating

  • Viral


Nucleic acids cell overview and core topics

UV Light Causes Pyrimidine Dimerization

  • Replication and gene expression are blocked


Nucleic acids cell overview and core topics

  • Chemical mutagens

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


Nucleic acids cell overview and core topics

  • Deaminating agents

    • Ex: Nitrous acid (HNO2)

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

    • Causes A-T to G-C transitions


Nucleic acids cell overview and core topics

  • Alkylating agents


Nucleic acids cell overview and core topics

  • Intercalating agents


Nucleic acids cell overview and core topics

  • Acridines

    • Intercalate in DNA, leading to insertion or deletion

    • The reading frame during translation is changed


Nucleic acids cell overview and core topics

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)


Nucleic acids cell overview and core topics

Do we has a quiz?


Nucleic acids cell overview and core topics

QUIZ

  • 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

RNA Transcription


Nucleic acids cell overview and core topics

Process of Transcription has four stages:

  • Binding of RNA polymerase at promoter sites

  • Initiation of polymerization

  • Chain elongation

  • Chain termination


Nucleic acids cell overview and core topics

Transcription (RNA Synthesis)

  • RNA Polymerases

    • Template (DNA)

    • Activated precursors (NTP)

    • Divalent metal ion (Mg2+ or Mn2+)

  • Mechanism is similar to DNA Synthesis


Nucleic acids cell overview and core topics

Reece R. Analysis of Genes and Genomes.2004. p47.

  • 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)


Nucleic acids cell overview and core topics

Start of Transcription

  • Promoter Sites

    • Where RNA Polymerase can indirectly bind


Nucleic acids cell overview and core topics

Preinitiation Complex (PIC)

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.


Nucleic acids cell overview and core topics

Termination of Transcription

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


Nucleic acids cell overview and core topics

Termination of Transcription

2. Rho termination = Rho protein, ρ


Nucleic acids cell overview and core topics

prokaryotes: transcription and translation happen in cytoplasm

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


Nucleic acids cell overview and core topics

  • 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


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

Introns & Exons

  • Introns

    • Intervening sequences

  • Exons

    • Expressed sequences


Nucleic acids cell overview and core topics

Splicing

Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs)

splicing occurs in the spliceosome!


Nucleic acids cell overview and core topics

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"


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

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


Translation protein synthesis

Central Dogma

Translation: Protein Synthesis


Nucleic acids cell overview and core topics

Translation

Starring three types of RNA

  • mRNA

  • tRNA

  • rRNA


Nucleic acids cell overview and core topics

Properties of mRNA

  • 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


Nucleic acids cell overview and core topics

Genetic Code

  • 3 nucleotides (codon) encode an amino acid

  • The code is nonoverlapping

  • The code has no punctuation


Nucleic acids cell overview and core topics

Synonyms

  • Different codons, same amino acid

  • Most differ by the last base

    • XYC & XYU

    • XYG & XYA

  • Minimizes the deleterious effect of mutation


Nucleic acids cell overview and core topics

Practice

  • 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.


Nucleic acids cell overview and core topics

Answers

  • (a) 5’ -UAACGGUACGAU-3’ .

  • (b) Leu-Pro-Ser-Asp-Trp-Met.


Nucleic acids cell overview and core topics

tRNA as Adaptor Molecules

  • Amino acid attachment site

  • Template recognition site

    • Anticodon

      • Recognizes codon in mRNA


Nucleic acids cell overview and core topics

tRNA as Adaptor Molecules


Nucleic acids cell overview and core topics

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.

  • Terminationoccurs when "stop codon" reached


Nucleic acids cell overview and core topics

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


Nucleic acids cell overview and core topics

Translation

  • Eukaryote START

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


Nucleic acids cell overview and core topics

Termination

  • Stop signals (UAA, UGA, UAG):

    • recognized by release factors (RFs)

    • hydrolysis of ester bond between polypeptide and tRNA


Nucleic acids cell overview and core topics

Reference:

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

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


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