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Introduction

  • All organisms use the same genetic code. Bacteria have the same genetic “language” as humans.
  • In the 1940s-50s, scientists were biased: they didn’t believe that a genetic language could only have 4 “letters”
  • A genetic language must be able to be
    • Accurately copied and passed on to the progeny
      • Mutation could result otherwise.
      • Like a photocopier: after many copies, it could become unclear.
      • Our copy process is composite: we pass the copy of the copy of the … of the copy to our children etc. It must be reliable
    • Readily accessed for the information that it contains
      • Don’t want a .exe file on a Mac
  • Two molecules were considered to be candidates for a genetic language: proteins and DNA

Proteins vs DNA

  • Proteins have great complexity and 20 monomer building blocks (amino acids)
  • DNA has a very regular structure and only 4 monomer blocks
  • Scientific bias: they thought protein was more likely
  • However, experimental data indicated that DNA might be the genetic material.

Hershey and Chase experiment – 1953

Using bacteriophage T2, a bacterial virus, they demonstrated that DNA is the genetic material. Phage = virus. Latches onto the surface of a cell and injects its genetic information inside the cell. The virus takes over the replication of the cell. They did it using two tests:

Test 1

  1. Virus was given radioactive DNA (phage grown with radioactive phosphorous-32: only present in DNA). The virus was allowed to infect the bacteria
  2. The solution was blended to shear the virus off the cell.
  3. This left the empty protein shell/coat of the virus in the extracellular solution, and the phage/bacteria hybrid DNA inside the cell
  4. Centrifugation separated the phage’s proteinaceous remains (fluid) from the bacteria (pellet).
  5. Radioactivity was detected only in the pellet

Test 2

  1. Virus was given radioactive protein (phage grown with radioactive sulfur-35: only present in protein). The virus was allowed to infect the bacteria.
  2. The solution was blended to shear the virus off the cell.
  3. This left the empty protein shell/coat of the virus in the extracellular solution, and the phage/bacteria hybrid DNA inside the cell
  4. Centrifugation separated the phage’s proteinaceous remains (fluid) from the bacteria (pellet).
  5. Radioactivity was detected only in the fluid.

Historical perspective

  • In 1953, we knew that
    • DNA is the genetic material
    • It has repeating sugar-phosphate units
    • It contains nucleotide bases: Cytosine, Guanine, Adenine and Thymine.
    • The base composition varies in different organisms
    • However, in every organism, A=T and G=C
    • The structure of DNA was unknown

Watson and Crick

  • April 1953, Watson and Crick published the structure of DNA based on molecular models using X-ray diffraction data from Rosalind Franklin
  • Purines and pyrimidines
Purines and pyrimidines
    • There are 4 bases: C, G, A, T
    • There are two purines, A and G
      • Big molecules
    • There are two pyrimidines, T and C
      • Small rings
    • They are made of C, N, O, H
    • Each has deoxyribose attached
    • The red highlighted atoms are important for hydrogen bonding.
    • Hydrogen bonding determines the helical structure of DNA.
    • The shapes of the molecules dictate the fact that C pairs with G and A pairs with T.
    • Purines (large) pair with pyrimidines (small) – otherwise there would be “bulges” and “dips” in the DNA helix. Big guy pairs to little guy.
Base pairs
    • A always pairs with T:
      • A is a purine, T is a pyrimidine
      • They both have 2 hydrogen bonding sites – they match
      • This means these pairings are a little bit weaker than G-C
    • G always pairs with C
      • G is a purine, C is a pyrimidine
      • They both have 3 hydrogen bonding sites – they match
      • This means these pairings are a little bit stronger than A-T – the cell exploits this in some ways
    • If you mix the bases together in an aqueous solution of neutral pH, these pairings will always take place.

Deoxyribonucleotide

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  • Ribose is a 5 carbon sugar with a ring structure.
  • “Deoxy” because the highlighted H is an OH in ribose – ribose is relatively oxidised
  • The base is at 1’, the phosphate is at 5’.

Double stranded DNA helix

  • DNA is an alpha helix - not a spiral
  • Alpha helix is defined by:
    • Put your thumb in the direction of the chain (5’ to 3’) and your fingers wrap in the direction that it goes
    • (A beta helix is the opposite direction of wrapping)
  • The backbone is made of phosphate-sugar-phosphate-…
  • The phosphate (5’) end of one deoxyribonucleotide polymerises with the 3’ OH on another deoxyribonucleotide
  • The two DNA strands are antiparallel: they always run in opposite directions with respect to the sugar-phosphate backbone.
  • Directionality: 5’ to 3’ ALWAYS

DNA replication

  • Both strands of DNA act as templates for the synthesis of new DNA strands
  • This is a semi-conservative process
  • The assembly of machinery for replication starts at multiple locations along the DNA, called “origins of replication” – bubbles of opened DNA (“replication bubbles”), out from which replication proceeds.
  • Helicases separate the two DNA strands and DNA polymerase “reads” the old strand and synthesises a new strand.
  • As the machinery moves along “reading” the old strand and synthesising the new one, the bubbles merge.
  • Multiple origins are needed so that all the DNA in a cell can be replicated in a short time
  • Human cells contain 6 billion base pairs, which are all copied within a few hours.
  • DNA polymerases insert the bases with great accuracy. If the base it’s carrying doesn’t fit, then the Phosphate bonds won’t break – this needs to happen for the polymerisation to occur. Hence this is an auto-check mechanism.
    • P bonds have lots of energy, which is used to catalyse the formation of the sugar-phosphate bond in the backbone of the DNA
    • The Phosphate on the 5’ of one deoxyribose binds to the OH on the 3’ of the nearby deoxyribose.

AZT diagram

  • AZT is an analogue of thymidine. It blocks DNA replication and is used to treat AIDS (HIV) infection. It contains an azido group (-N-N=N-) instead of an OH group, which blocks the next monomer unit from polymerising. It is the first drug to successfully treat AIDS.
    • Note nucleosides = NB + pentose = {deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine, deoxyuridine}
    • NB = {adenine, guanine, thymine, cytosine, uracil}
  • Although we don’t have a strikingly large number of genes in our genome compared to other organisms, the way we access and use the genes is more diverse, giving more complexity.
  • The human genome:
    • 3.2 billion base pairs, less than 5% are coding genes, estimated 30,000 genes.
      • Note that the other 95% is not all junk (it includes control/regulation information that we don’t understand fully)
    • The average gene has 27,000 base pairs (range from 1,000 to 2.4 million base pairs)
    • Almost all (99.9%) of the genome is the same in all people.
    • Genes are not evenly distributed (chr 1 has 2968 genes, Y chr has only 231)
    • The functions of many genes are still not known.

Protein synthesis

Process whereby DNA encodes for the production of amino acids and proteins. This process can be divided into two parts: Transcription and Translation

Transcription

  • Before the synthesis of a protein begins, the corresponding RNA molecule is produced by RNA transcription
  • One strand of the DNA double helix is used as a template by the RNA polymerase to synthesize a messenger RNA (mRNA)
  • This mRNA migrates from the nucleus to the cytoplasm. During this step, mRNA goes through different types of maturation including one called splicing when the non-coding sequences are eliminated
  • The coding mRNA sequence can be described as a unit of three nucleotides called a codon

Translation

  • The ribosome binds to the mRNA at the start codon (AUG) that is recognized only by the initiator tRN.
  • The ribosome proceeds to the elongation phase of protein synthesis. During this stage, complexes, composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon
  • The ribosome moves from codon to codon along the mRNA
  • Amino acids are added one by one, translated into polypeptidic sequences dictated by DNA and represented by mRNA
  • At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome
  • One specific amino acid can correspond to more than codon. The genetic code is said to be degenerate.

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