Biol1010

Chapter 12: DNA structure and functions

Questions:
 Describe and explain Griffith's transformation experiment.
 Describe and explain Chargaff’s findings.
 What is Chargaff’s rule?
 Describe and explain Avery and colleague’s findings.
 Describe Hershey and Chase’s experiment.
 Define transformation.
 Describe the structure of a nucleotide.
 Describe the discoveries of Franklin, Watson and Crick.
 What was revealed by the X-ray diffraction experiment by Franklin?
 What is the Watson-Crick Model?
 Compare prokaryotic DNA structure to eukaryotic and explain how this makes
DNA replication look different in the two types of cells.
 How is prokaryotic DNA replication similar to eukaryotic?
 How is prokaryotic DNA replication different from eukaryotic?
 How can the structure of DNA explain DNA replication?
 What are telomeres and why are they important?
 Explain the process of DNA replication. Use the terms origin or replication, DNA
polymerase, leading strand, lagging strand, Okazaki fragments, replication fork,
RNA polymerase, DNA helicase, DNA ligase.
 What are the three major steps of DNA replication?
 What are the enzymes involved in DNA replication and what do they do?
 What does it mean that DNA replication is semi-conservative?
 DNA is replicated 5' - 3'. What do the numbers refer to?
 Why are DNA strands anti-parallel?
 Why are Okazaki fragments formed on the lagging strand?
 What is the central dogma?
 What does it mean that the genetic code is universal?
 How is the existence of a universal genetic code explained by the theory of
evolution?
 What does it mean that the genetic code is degenerate?
 What does it mean that the genetic code is unambiguous?
 What are the different types of RNA molecules in the cell, and what are their
functions?
 Explain how proteins are made in the cell, starting from DNA.
 What are the different parts of a mature mRNA?
 Explain the Poly-A tail and 5’cap in the mature mRNA.
 Describe mRNA processing, using the terms cap, poly-A tail, intron, exon,
splicing, spliceosome.
 Why are exons exported and introns stay inside the nucleus?
 What is a start codon?
 What is a stop codon?

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  Describe the structure and important parts of the tRNA.
 Explain how tRNA works in the process of protein synthesis.
 Describe the different steps of translation.
 Describe the structure and sites of the ribosome.

Notes:
Frederick Griffith's experiments indicated the existence of genetic material. Follow-up
experiments by others showed the existence of DNA as the genetic material.
Today, genes can be isolated and transferred from one organism to another. The resulting
organism is called a genetically modified organism (GMO).
Transformation is the transfer of genetic material (genes) between organisms.
Chargaff's rules:
The relative A,T,C and G amounts vary from species to species, but is constant
within a species.
In each species, the amount of A=T and C=G

DNA nucleotides contain a deoxyribose sugar, a purine (A/G) and pyrimidine (T/C)
nitrogenous base and a phosphate group. Nucleotides are joined together by DNA
polymerase into helices. A double helix is generated by complementary base pairing.
Watson and Crick described the structure of DNA - the double helix
We can understand replication, base-pairing and the genetic code of DNA by
understanding the basic structure of DNA.

DNA double helixes can be separated down the middle (breaking of the A-T and G-C
hydrogen bonds).

DNA replication
 DNA replication starts at the origin of replication.
 Both DNA strands are templates for DNA replication, resulting in
semiconservative replication
 The steps of DNA replication are: Unwinding and unzipping by DNA helicases,
Complementary base pairing by DNA polymerase, Joining of DNA nucleotides in
the new strand by DNA ligase.
 Helicases unwind the DNA strands and expose the bases.
 The bases will be matched by complementary base-pairing (old strands are used
as templates for the new strands) by DNA polymerase. A-T, G-C.
 Due to the nature of the DNA polymerases, replication can only start after an
RNA polymerase has made a starting duplex for the DNA polymerase (primer).
DNA polymerases cannot start DNA replication on their own.
 Due to the nature of DNA replication, replication of a DNA strand can only occur
in one direction (5'-3'). 5’ vs. 3’ indicate the position of the carbon in the
deoxyribose (sugar) of the nucleotide. It is really an indication of the orientation
of the sugar in the strand.
 The strands of a DNA duplex are therefore always anti-parallel.
 Replication is carried out differently on the "leading" and "lagging" DNA strand.
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  Okazaki fragments (made by DNA polymerase) on the lagging strand form, since
replication is going in the direction away from the origin of replication on this
strand. The fragments are joined by DNA ligase. The replication is discontinuous
on the lagging strand.
 On the leading strand, the DNA polymerase can follow the DNA helicase and the
DNA replication is therefore continuous.
 DNA polymerase cannot replicate the very last end of a linear chromosome.
 Linear chromosomes of Eukaryotes are protected by the telomere sequences.
Those sequences are repetitive DNA sequences. The telomeres shorten with cell
divisions, and contribute to inhibiting uncontrolled cell growth (cancer).

Prokaryotes and Eukaryotes replicate their DNA according to the same principles, but
Prokaryotes have one circular chromosome, and Eukaryotes have linear chromosomes.
Replication starts at the origin of replication – one in Prokaryotes and several on each
chromosome in Eukaryotes. Replication bubbles with two replication forks each form and
spread along the chromosome/s.

DNA polymerase proofreads and corrects mistakes in replication. DNA repair enzymes
fix errors and DNA strand breaks. Those actions contribute to low DNA mutation rates.

There are several types of RNA (ribonucleic acid).

Messenger (m)RNA (codes for protein)
Transfer (t)RNA (transfers amino acids to the ribosome)
Ribosomal (r)RNA (part of the ribosome)
microRNAs (ex. miRNA, participates in control of gene expression, snRNAs are core
components of the spliceosome)

The genetic code is a triplet of nucleotides that code for an amino acid.
Therefore, a protein that is 100 amino acids long would be coded by 300 nucleotides, or
100 codons.
The genetic code is universal – it is the same for virtually all organisms.
It is degenerate – the first two nucleotides are generally the same for a particular amino
acid, and the third nucleotide differs.
The genetic code is unambiguous: One codon (for instance UUU) codes for one specific
amino acid (phenylalanine).
There are codons that mean “start” (AUG) and “stop” (UAA, UGA, UAG).

GeneExpression (Protein Synthesis)

Transcription: Formation of mRNA with DNA as a templat, by RNApolymerase. Takes
place in the nucleus.
 Initiation: RNA polymerase initiates transcription at the gene’s promoter
(beginning of a gene).
 Elongation: mRNA is produced by complementary basepairing. RNA polymerase
generates pre-mRNA from the coding template DNA strand of the gene.
 Termination: A stop codon is reached and RNA polymerase detaches. Pre-mRNA
is now synthesized.

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mRNA processing:
Precursor mRNA is formed in transcription. It is processed in the nucleus prior to export
into the cell cytoplasm. A pre mRNA has introns and exons. The introns are excised
during splicing in the spliceosome. A cap is added to the 5’ end (guides to the start site
for the ribosome) and a PolyA-tail is added to the 3’ end (protects from degradation).

Translation: Formation of a protein with mRNA as a template. Occurs in the cytoplasm.

The ribosome has two subunits and an E, P and A site. The tRNA has an anticodon that is
complementary to the mRNA codons. The tRNA carries one specific amino acid – the
one that is specified by the codon (the genetic code). tRNA is charged with a new amino
acid by the enzyme tRNA synthetase.
 Initiation: Initiation factors assemble the small ribosomal subunit, mRNA,
initiator tRNA (codes for methionine and has anti-codon for AUG. Large
subunit joins. Eukaryotic initiation is more complex than that in prokaryotes.
 Elongation: new tRNA binds to the A-site. A polypeptide is created by
transferring and joining the aminoacid chain to the A-site tRNA. The ribosome
moves one step forward (translocation), and the tRNA with the attached
polypeptide is now in the P site. The empty tRNA is now in the E site and will
exit from there.
 Termination: When a stop-codon occurs in the A site, a release factor will bind
there, and the ribosome will disassemble. The protein has new been generated. It
will be folded and attain its function in the cell.

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