Molecular Biology

MODULE 2 - MOLECULAR BIOLOGY

  • DNA and the genetic code

  • DNA replication

  • Transcription

  • RNA processing

  • Translation

  • Regulation of gene expression

  • Practical applications of molecular biology

DNA AND THE GENETIC CODE; DNA REPLICATION

Reading

  • Campbell: The molecular basis of inheritance

Lecture Topics

  • Early evidence that DNA is the carrier of genetic information

  • Brief review of DNA structure

  • Biochemistry of DNA replication

  • Early evidence that genes encode proteins

THE GENETIC BLUEPRINT

  • Contains the "instructions" or "parts list" for creating a cell

  • 3 fundamental properties:

    • stores information

    • can be accurately copied & transmitted to progeny

    • has the capacity to change (mutate)

  • Chemical nature of the genetic blueprint?

EARLY EVIDENCE THAT DNA CARRIES GENETIC INFORMATION

Classic experiment of Frederick Griffith (1928)

  • Studied pneumonia caused by a bacterium: Streptococcus pneumoniae

  • Two classes of bacteria:

    • smooth (S) = virulent strain (kills mice)

    • rough (R) = avirulent (doesn’t kill mice)

    • both traits inherited

  • S strain makes protective polysaccharide capsule

  • R strain lacks capsule - killed by mouse immune system

TRANSFORMATION

  • Genetic information for virulence transferred from dead S cells to live R cells

  • Frederick Griffith found that heat-treatment didn't destroy the "transforming" activity.

  • Other scientists showed that the transforming activity was bacterial DNA, not proteins or other molecules

EXPERIMENT BY ALFRED HERSHEY AND MARTHA CHASE (1952)

  • Studied bacteriophage T2; a virus that infects E. coli

  • Virus injects genetic blueprint into cell and uses cell’s biosynthetic machinery to make more viruses

  • Question: Which molecule carries the genetic blueprint for making a phage: DNA or protein?

"WARING BLENDER" EXPERIMENT OF HERSHEY AND CHASE

  • Batch 1: Radioactive sulfur (35S^{35}S) in phage protein

    1. Labeled phages infect cells.

    2. Agitation frees outside phage parts from cells.

    3. Centrifuged cells form a pellet.

    4. Radioactivity (phage protein) found in liquid.

  • Batch 2: Radioactive phosphorus (32P^{32}P) in phage DNA

    1. Labeled phages infect cells.

    2. Agitation frees outside phage parts from cells.

    3. Centrifuged cells form a pellet.

    4. Radioactivity (phage DNA) found in pellet.

  • Conclusion: DNA - not protein - carries genetic information

DNA AS A CARRIER OF GENETIC INFORMATION

  • DNA is a polymer of only 4 building blocks (A, C, G and T)

  • How does this simple molecule carry genetic information?

DNA STRUCTURE DETERMINATION

  • James Watson, Francis Crick, Maurice Wilkins and Rosalind Franklin (first three shared Nobel prize in 1962)

  • Next big advance (1953): DNA structure determined using X-ray crystallography

  • Two anti-parallel strands of DNA from double helix.

  • Hydrophilic phosphate groups and deoxyribose are exposed.

  • N-bases face inward and interact via H-bonds (A with T; G with C)

DNA STRANDS

  • Each DNA strand has 5’ and 3’ ends

FUNCTION OF DNA AS THE GENETIC BLUEPRINT

  1. How does it store information? Sequence of bases.

  2. How does it mutate? Change sequence of bases.

  3. How is it accurately copied (replicated) prior to cell division? Two DNA strands are complementary. The sequence of each strand contains the information needed to make a perfect copy of the other strand.

DNA REPLICATION

  • DNA replication is semi-conservative

BIOCHEMISTRY OF DNA REPLICATION

  • Very accurate and rapid (50-500 bases per second)

  • DNA synthesized by DNA polymerase

  • 6 billion base pairs in human DNA, copied in a few hours

  • <1 mistake per billion bases!

  • Must ensure that DNA is copied once (and only once) per cell division

ORIGIN OF REPLICATION

  • Process begins at origin of replication

DNA REPLICATION DETAILS

  • Complementary DNA strands pulled apart by DNA helicase

  • DNA replication is bi-directional: two replication forks per bubble

  • Prokaryotic chromosomes have one origin

  • Eukaryotic chromosomes have many origins

PROTEINS INVOLVED IN DNA REPLICATION

  • Complementary DNA strands pulled apart by DNA helicase

  • Single-stranded DNA-binding proteins keep helix from reforming

  • Topoisomerases keep DNA from getting tangled up as it is unwound

DNA POLYMERASE

  • DNA polymerase only adds nucleotides to the 3'OH of the growing strand

  • Cannot initiate DNA synthesis without a "primer": nucleic acid containing a 3'OH to which next nucleotide can be added

TEMPLATE, PRIMER AND COMPLEMENTARY STRAND

  • Newly synthesized DNA is complementary to the template strand

  • Must understand the terms "primer", "template strand" and "complementary strand" and how they are related to the 5' and 3' ends of nucleic acid polymers!

DNA POLYMERASES

  • DNA polymerases need a template and a primer to synthesize DNA

  • High-energy phosphodiester bond cleaved as the next nucleotide added to 3'OH at end of growing chain. Provides the energy that drives the reaction

PRIMER NEED

  • If DNA polymerase needs a primer (3'OH ), how does DNA synthesis begin at the origin?

  • "Primase" makes a short RNA primer to start DNA synthesis.

LEADING STRAND SYNTHESIS

  • Allows the synthesis of the "leading strand" of DNA

LAGGING STRAND SYNTHESIS

  • How is the other ("lagging") strand of DNA synthesized?

  • Primase makes one RNA primer for the leading strand

  • Must make additional primers for lagging strand as helix opens

  • Priming occurs once for leading strand, many times for lagging strand

  • Lagging strand is made in short pieces of DNA = "Okazaki fragments” (100’s of nucleotides long)

LEADING AND LAGGING STRANDS

  • Overview showing the leading and lagging strands during DNA replication

COMPLETING LAGGING STRAND SYNTHESIS

  • Primers must be removed and Okazaki fragments connected to complete the synthesis of the lagging strand

  • Additional enzymes that carry out these steps: DNA polymerase I and DNA ligase

ENZYMES INVOLVED IN DNA REPLICATION

  • Primase: synthesizes RNA primer, primes once for leading strand; many times for lagging strand

  • DNA polymerase III: adds nucleotides to 3’OH of RNA primer

  • DNA polymerase I replaces the RNA primers with DNA

  • DNA ligase joins the Okazaki fragments to form a continuous DNA strand.

REPLACING PRIMERS

  • Primers must be replaced with DNA and Okazaki fragments connected to complete the synthesis of the lagging strand

PROTEINS WORKING TOGETHER

  • Illustrated representation of the Primase, DNA Pol III, DNA Pol I, and DNA ligase working together during synthesis of leading and lagging strands during DNA replication.

ACCURACY OF DNA REPLICATION

  • 6 billion base pairs in human DNA, copied in a few hours

  • Errors cause mutations!

  • Replication is very accurate: <1 mistake per billion bases!

  • DNA polymerase “proofreads” its works and corrects errors as soon as they are made

  • Multiple mechanisms exist to repair environmental damage to DNA

KEY CONCEPTS ABOUT THE BIOCHEMISTRY OF DNA REPLICATION

  • Understand replication origins, bubbles and forks

  • Be able to draw nucleotides, the DNA polymer and a DNA double helix

  • Understand how nucleotides are added to the growing polymer

  • Understand the similarities and differences between the synthesis of the leading and lagging strands

  • Understand the functions of the following proteins: DNA helicase, topoisomerase, ssDNA-binding proteins, primase, DNA Pol III, DNA Pol I, DNA ligase

  • Be able to draw out replication process at the level of detail presented in lecture

MOLECULAR BIOLOGY AND GENETICS

  • A gene is a unit of heredity that is passed from parent to offspring

  • Each gene specifies one or more traits of an organism

  • Each gene corresponds to a specific regions of DNA

EARLY EVIDENCE SUGGESTING THAT GENES ENCODE PROTEINS

  • 1909: Archibald Garrod published Inborn Errors of Metabolism

    • some inherited disorders caused by defects in an enzyme

    • alkaptonuria: alkapton accumulates due to lack of an enzyme, causing patients to have black urine

    • albinism: lack of the enzyme that makes melanin, a skin pigment

    • proposed that defect in a "gene" is associated with defect in an enzyme

  • 1940's: George Beadle and Edward Tatum studied three different Neurospora strains that are unable to grow without arginine

    • Each strain lacked one of three enzymes required for arginine biosynthesis

    • Led them to propose the "one gene – one enzyme" hypothesis

GENE TO PROTEIN

  • How does a gene (DNA) direct the synthesis of a protein?

  • Genetic information of DNA stored in sequence of bases.

  • Sequence of bases in DNA specifies primary sequence (amino acid sequence) of protein

  • Cell must convert base sequence à amino acid sequence

  • Conversion is not direct! Occurs through RNA intermediate = mRNA (messenger RNA)

GENETIC LANGUAGE

  • DNA and RNA share the same "genetic language" = sequence of bases

  • Sequence of nucleotides in a gene (DNA) is "transcribed" into sequence of nucleotides in an RNA molecule

  • Sequence of nucleotides in an RNA is "translated" into sequence of amino acids in a polypeptide

CENTRAL DOGMA

  • transcription translation
    DNA --> RNA --> protein

  • RNA polymerase ribosomes

  • Exceptions to central dogma are rare

NUCLEOTIDE SEQUENCE

  • 4 nucleotides (A, C, G, T or U) must specify 20 amino acids

    • 16 possible doublets (AT, TG, etc.)

    • 64 possible triplets (ATG, CCC, etc).

    • Need at least 3 bases to encode all amino acids

  • Three bases that specify an amino acid = a CODON

  • The codons for each amino acid determined in the 1960's

  • 3000 bases of DNA encode a protein with 1000 amino acids.

  • 1 Mb (megabase) = 1000 kb (kilobase) = 1,000,000 bases.

  • How does a nucleotide sequence (DNA or RNA) specify the amino acid sequence of a protein?

GENETIC CODE

  1. The genetic code is redundant: different codons can specify the same amino acid.

    • e.g. both GUU and GUC encode valine

  2. The genetic code is not ambiguous: no codon specifies more than one amino acid.

    • can predict exact protein sequence from nucleotide sequence

  3. The genetic code is universal: same in bacteria and humans.

    • very important for biotechnology industry.

READING FRAMES

  • In theory, any base sequence can be read in three different "frames"

  • Special codons must determine where "translation" begins and ends!

    • AUG = start (initiation) codon

    • UAA, UAG, UGA = stop (termination) codons; don't encode amino acids.

  • The following RNA would be translated as shown:

    • 5' UUUUAUGUCUAACGAAUAAAAUAA3'

    • M S K E

  • A stretch of bases uninterrupted by termination codons is called an "open-reading frame" or ORF.

CODON TABLE

  • A codon table will be provided to use on exam

MESSENGER RNA (mRNA)

  • Anatomy of a typical messenger RNA (mRNA)

    • 5'UTR (untranslated region)

    • AUG (start codon)

    • ORF (open reading frame)

    • STOP (stop codon)

    • 3'UTR (untranslated region)Translation protein N C