DNA Structure and Replication

1
DNA Structure and Replication
2

• DNA structure
• The DNA double helix was identified by Watson,
  Crick, and Franklin in the late 1950's as being
  the most likely structure
• for DNA.
• The primary features of the structure
• Its a double helix (two strands wrapped around
  each other)
• Its composed of repeating chemical units called
  nucleotides
• The sugar-phosphate backbone is on the outside
• The phosphate groups give the whole molecule a
  negative charge

3
http//faculty.virginia.edu/bio201/lecture/DNA20s
tructure.jpg
4
http//www.umass.edu/microbio/rasmol/helix-2.htm
(emartz_at_microbio.umass.edu)
5
http//faculty.virginia.edu/bio201/lecture/base20
pairs.jpg
6

• Other important features
• The nucleotides from each strand are bonded
  together with hydrogen bonds
• Theres a polarity to the axes. The sequence of
  a nucleic acid is customarily read from 5' to 3'.
  
• Because of the complementarity of the base
  pairing, DNAs structure creates a redundancy in
  the code.

7
Bases are stacked on top of one another for
increased stability
http//www.umass.edu/microbio/rasmol/dna-2bp.htm
8

• The human genome contains about 3 billion
  nucleotide pairs organized as 23 chromosomes
  pairs.
• If uncoiled, the DNA contained by each of those
  chromosomes would measure between about 2 - 9 cm.
  
• Chromosomal DNA is packaged into a compact
  chromatin structure with the help of specialized
  proteins called histones.
• The fundamental packing unit is known as a
  nucleosome.
• 1) The DNA double helix wraps around a central
  core of eight histone protein molecules (an
  octamer) to form a single nucleosome.
• 2) A second histone (H1 in the illustration)
  fastens the DNA to the nucleosome core.
• 3) The total mass of this complex is about
  100,000 daltons.

9
Nucleosome Subunit of chromatin composed of a
short length of DNA wrapped around a core of
histone proteins. http//www.accessexcellence.or
g/AB/GG/nucleosome.html
10
http//www.accessexcellence.org/AB/GG/chroma_packg
.html
11
Comparative Sequence Sizes
(Bases) Yeast chromosome 3
350,000 Escherichia coli
(bacterium) genome 4.6
Million Largest yeast chromosome now mapped
5.8 Million Entire yeast genome
15 Million Smallest human
chromosome (Y) 50
Million Largest human chromosome (1)
250 Million Entire human genome
3 Billion
12
Physical Map
Genetic Map
http//www.accessexcellence.org/AB/GG/sequences.ht
ml
13
DNA Replication There are three possible ways
that DNA could have been replicated.
Conservative replication would leave intact the
original DNA molecule and generate a completely
new molecule. Dispersive replication
would produce two DNA molecules with sections of
both old and new DNA interspersed along each
strand. Semiconservative replication
would produce molecules with both old and new
DNA, but each molecule would be composed of one
old strand and one new one.
14
http//www.accessexcellence.org/AB/GG/possible.htm
l
15
When DNA replicates, many different proteins work
together to accomplish the following steps
1.The two parent strands are unwound with the
help of DNA helicases. 2. Single stranded DNA
binding proteins attach to the unwound strands,
preventing them from winding back
together. 3.The strands are held in position,
binding easily to DNA polymerase, which catalyzes
the elongation of the leading and lagging
strands. 4.While the DNA polymerase on the
leading strand can operate in a continuous
fashion, RNA primers are needed on the lagging
strand to facilitate synthesis of Okazaki
fragments. DNA primase, which is one of several
polypeptides bound together in a group called
primosomes, helps to build the primer. 5.Finally,
each new Okazaki fragment is attached to the
completed portion of the lagging strand in a
reaction catalyzed by DNA ligase.
16
http//www.accessexcellence.org/AB/GG/collaboratio
n.html
17

• Ribonucleic Acids (RNAs)
• There are four types of RNA
• transfer RNA tRNA
• messenger RNA mRNA
• ribosomal RNA rRNA
• small nuclear RNA snRNA
• RNA is different chemically from DNA
• Both RNA and DNA form long, unbranched
  polynucleotide chains but...
• In RNA, the nucleotides are specifically
  ribonucleotide monophosphates.
• In DNA, the nucleotides are specifically
  deoxyribonucleotide monophates.

18
mRNA
SnRNA

tRNA RNA mRNA
http//www.accessexcellence.org/AB/GG/central.html
19
http//www.accessexcellence.org/AB/GG/rna2.html
20
The differences in chemical composition and
structure creates important differences between
DNA and RNA 1) The chemical composition of the
DNA backbone make it more stable than RNA. 2)
RNA usually forms intramolecular base pairs 3)
The information carried by RNA is not redundant

21

• Messenger RNA (mRNA)
• Messenger or mRNA is a copy of the information
  carried by a gene on the DNA.
• Its the product of the nuclear process called
  transcription.
• The role of mRNA is to move the information
  contained in the DNA to the translation machinery
  in the cytoplasm
• mRNAs are heterogeneous in size and sequence
  because they are specific to each gene.

22
Messenger RNA (mRNA)
http//www.biochem.uwo.ca/meds/medna/mRNA.html
23
Transfer RNA (tRNA)

• Stem-Loop Diagram of a tRNA
• Each tRNA has an acceptor stem. This is the
  site at which a specific amino acid is attached
  by an amino-acyl-tRNA synthase.
• The anticodon reads the information in a mRNA
  sequence by base pairing.

http//www.biochem.uwo.ca/meds/medna/tRNA.html
24
A 3-D model of a yeast tRNA molecule which can
code for Serine (Ser). The gray and orange are
the sugar - phosphate backbone.
http//www.biochem.uwo.ca/meds/medna/tRNA.html
25

• rRNA and ribosome synthesis
• Ribosomal RNA (rRNA) is a component of the
  ribosomes.
• Eukaryotic ribosomes contain four different rRNA
  molecules
• 18 s, 5.8 s, 28 s, and 5 s rRNA.
• Three of the rRNA molecules are synthesized in
  the nucleolus, and one is synthesized elsewhere.
• rRNA molecules are extremely abundant.
• They make up at least 80 of the RNA molecules
  found in a typical eukaryotic cell.
• http//www.biochem.uwo.ca/meds/medna/rRNA.html

26

• Synthesis of the three nucleolar rRNA molecules
  is unusual because they are made on one primary
  transcript that is chopped up into three mature
  rRNA molecules.
• These rRNA molecules and the 5 s rRNA combine
• with the ribosomal proteins in the nucleolus to
  form
• pre 40 s and pre 60 s ribosomal subunits.
• These pre-subunits are exported to the nucleus
  where
• they mature and assume their role in protein
  synthesis.
• http//www.biochem.uwo.ca/meds/medna/rRNA.html

27
http//www.biochem.uwo.ca/meds/medna/rRNA.html
28

• small nuclear RNA (snRNAs)
• These RNA molecules are important in a number of
  processes including RNA splicing (removal of the
  introns from hnRNA) and maintenance of the
  telomeres, or chromosome ends.
• They are always found associated with specific
  proteins and the complexes are referred to as
  small nuclear ribonucleoproteins (SnRNP) or
  sometimes as snurps.
• Antibodies against snurps are found in a number
  of autoimmune diseases (e.g. lupus)

29
http//acs.tamu.edu/ellison/gene.htm
30
Gene Expression
31
http//www.accessexcellence.org/AB/GG/exon2.html
32
mRNA is not made directly in a eukaryotic
cell. 1) It is transcribed as heterogeneous
nuclear RNA (hnRNA) in the nucleus. 2) hnRNA
contains introns and exons. 3) The introns are
removed by RNA splicing (by snurps) leaving the
exons joined together. 4) In some cases,
individual nucleotides can be added in the middle
of the mRNA sequence by a process called RNA
editing. hnRNA and mRNA are never found free in
the cell - like DNA, they are bound by cations
and proteins. These complexes are termed
ribonucleoproteins or RNPs. http//www.biochem.
uwo.ca/meds/medna/mRNA.html
33

• mRNA always has a 5 ' cap composed of a 5' to 5'
  triphosphate linkage between two modified
  nucleotides a 7-methylguanosine and a 2 '
  O-methyl purine.
• This cap serves to identify this RNA molecule as
  an mRNA to the translational machinery.
• In addition, most mRNA molecules contain a
  poly-adenosine tail at the 3' end.
• Both the 5' cap and the 3' tail are added after
  the RNA is transcribed and contribute to the
  stability of the mRNA in the cell.

34
Messenger RNA (mRNA)
http//www.biochem.uwo.ca/meds/medna/mRNA.html
35
http//www.accessexcellence.org/AB/GG/RNA_trans.ht
ml
36
In order to begin transcription, RNA polymerase
requires a number of general transcription
factors (called TFIIA, TFIIB, and so on). (A)
The promoter contains a DNA sequence called the
TATA box, which is located 25 nucleotides away
from the site where transcription is
initiated. (B) The TATA box is recognized and
bound by transcription factor TFIID, which then
enables the adjacent binding of TFIIB (C). For
simplicity the DNA distortion produced by the
binding of TFIID is not shown. (D) The rest of
the general transcription factors as well as the
RNA polymerase itself assemble at the promoter.
(E) TFIIH then uses ATP to phosphorylate RNA
polymerase II, changing its conformation so that
the polymerase is released from the complex and
is able to start transcribing. As shown, the
site of phosphorylation is a long polypeptide
tail that extends from the polymerase
molecule.The exact order in which the general
transcription factors assemble on the promoter is
not known. http//www.accessexcellence.org/AB/G
G/RNA_trans.html
37
http//www.accessexcellence.org/AB/GG/repressProt.
html
38
Regulation of gene expression by multiple
regulatory proteins
http//www.accessexcellence.org/AB/GG/regul_Prot.h
tml
39
Beta - globin gene expression

• Here are some of the gene regulatory proteins
  thought to control expression during red blood
  cell development.
• CP1 is found in many types of cells
• GATA-1 are present in only a few types of cells,
  including red blood cell precursors, and are
  therefore thought to the cell-type specificity of
  beta-globin gene expression.
• The bidirectional arrows indicate where there is
  competitive binding.
• (Adapted from B. Emerson, In Gene Expression
  General and Cell-Type Specific (M. Karin, ed.),
  pp. 116-161. Boston Birkhauser, 1993.)
  http//www.accessexcellence.org/AB/GG/B_globin.htm
  l

40
A hypothetical example of how a few different
regulatory proteins can result in many cell
types. In this hypothetical example, a
decision to make a new gene regulatory protein
is made after each cell division. Each of these
hypothetical cell types would then express
different genes, as dictated by the combination
of gene regulatory proteins that are present
within it.
41
RNA processing Splicing
42

• small nuclear RNA(snRNAs)
• These RNA molecules are important in a number of
  processes including RNA splicing (removal of the
  introns from hnRNA) and maintenance of the
  telomeres, or chromosome ends.
• They are always found associated with specific
  proteins and the complexes are referred to as
  small nuclear ribonucleoproteins (SnRNP) or
  sometimes as snurps.
• Antibodies against snurps are found in a number
  of autoimmune diseases (e.g. lupus)

43
http//acs.tamu.edu/ellison/gene.htm
44
http//acs.tamu.edu/ellison/gene.htm
45
http//acs.tamu.edu/ellison/gene.htm
46
http//acs.tamu.edu/ellison/gene.htm
47
http//acs.tamu.edu/ellison/gene.htm
48
http//acs.tamu.edu/ellison/gene.htm
49
Translation mRNA ---gt protein
50
http//www.accessexcellence.org/AB/GG/protein_synt
hesis.html
51
http//www.accessexcellence.org/AB/GG/exon2.html
52
http//www.accessexcellence.org/AB/GG/genetic.html
53
http//esg-www.mit.edu8001/esgbio/dogma/trl.html
54
http//esg-www.mit.edu8001/esgbio/dogma/trl.html
55
Protein bond formation
56
The Complete Atomic Structure of the Large
Ribosomal Subunit at 2.4 Å Resolution Nenad
Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore,
and Thomas A. Steitz Science 2000 August
11 289 905-920. (in Research Articles)
57
Proteins are orange and the rRNA is gray
58
(No Transcript)
59
Translation occurs in three stages 1)
Initiation The mature mRNA, the small ribosomal
subunit, and a tRNA that carries the first amino
acid (usually met) form a complex. The binding of
the big ribosomal subunit completes the
initiation stage. 2) Elongation The successive
binding of two charged-tRNA molecules into the P
and A sites of the ribosomes and creation of
peptide bonds bonds. Elongation continues until
the ribosome reaches a stop codon. 3)
Termination Once the stop codon is encounters
the translational complex breaks apart.
60
The polypeptide chain has several levels of
structure 1) Primary structure The sequence of
amino acids 2) Secondary structure Regular
interactions between R groups of the amino acids
(alpha helix, beta-pleated sheets, random
coiling) 3) Tertiary structure The final folding
configuration of the polypeptide chain 4)
Quarternary structure Functional structure that
is formed by the joining together of multiple
proteins (e.g. hemoglobin alpha and beta chains)
61
There are many control check points in protein
production
http//www.accessexcellence.org/AB/GG/control_Expr
ess.html