Unit IV- DNA, RNA, and
Protein Synthesis
Importance and Structure
of DNA: Deoxyribo-Nucleic Acid
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Historical Review:
1900’s – Morgan’s studies with fruit
flies showed that genes were located
on chromosomes and chromosomes
consisted of protein and DNA
 1952- Hershey-Chase demonstrated
that DNA (not protein) was the genetic
material of a viral phage
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Figure 16.2a The Hershey-Chase experiment: phages
Figure 16.2b The Hershey-Chase experiment
Phages Infecting a bacterium
Figure 16.1 Transformation of bacteria – Griffith (and later Avery, McCarty and
MacLeod)
The Structure of DNA
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Nucleotide monomers:
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Phosphate
Pentose Sugar (C5) –
Deoxyribose Sugar
Organic Nitrogen Base :
•
•
•
•
Cytosine (C)
Adenine (A)
Guanine (G)
Thymine (T)
Figure 16.3 The structure of a DNA stand
Structure of DNA cont’
Figure 16.3 The structure of a DNA stand
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Polynucleotide chain
with linkage via
phosphates to next
sugar, with nitrogen
base away from
backbone of
Phos-sugar-phos-sugar
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Dehydration synthesis
Beginning of the 1950’s several labs
were studying the structure of DNA
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Maurice Wilkins & Rosalind Franklin
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X-ray crystallography: x-rays pass
through pure DNA and defraction of xrays were then examined on film
James Watson and Francis Crick did
not have the expertise of Franklin
and were without proper photos
until………
Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA
Watson and Crick
Figure 16.0 Watson and Crick
Figure 16.0x James Watson
April 1953 – Classical one page
paper in Nature by Watson and
Crick
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A double helix – 2 polynucleotide strands
Sugar-phosphate chains of each strand
are like the side ropes of a rope ladder
Paris of nitrogen bases, one from each
strand, form the rungs or steps
The ladder forms a twist every 10 bases
(all from x-ray studies!)
Figure 16.5 The double helix
Internal Structure of DNA: Purine and pyridimine?
REMEMBER X-RAY DATA
Confirms Erwin Chargaff’s Rules
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# of Adenine = to #
of thymine
# of guanine equal to
# of cytosine
This dictates the
combinations of Nbases that form
steps/rungs
Does not restrict the
sequence of bases
along each DNA
strand
Replication/Duplication of DNA
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Due to complimentary base paring – one
strand of DNA determines the sequence
of the other strand
Therefore, each strand of double stranded
DNA acts as a template
The double helix first unwinds –
controlled by enzymes –and uses new
nucleotides that are free in the nucleus to
copy a complimentary strand off the
original DNA strand
Figure 16.7 A model for DNA replication: the basic concept (Layer 1)
Figure 16.7 A model for DNA replication: the basic concept (Layer 2)
Figure 16.7 A model for DNA replication: the basic concept (Layer 3)
Figure 16.7 A model for DNA replication: the basic concept (Layer 4)
Information storage in DNA
The 4 nitrogenous bases are the
“alphabet” or code for all the traits
the organism possesses
 Different genes or traits vary the
sequence and length of the bases
 ATTTCGGAC vs. GGGATTCTAG vs.
GATC
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There are a series of enzymes that
control DNA replication – enzymes
which:
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Uncoil the original double helix strand via
a helicase
Single-strand binding protein keeps helix
apart so replication can start
Prime an area to start replication –
primase except it adds RNA nucleotides at
first
Polymerase to join individual nucleotides
(dehydration synthesis)
Ligases to join short segments
Figure 16.8 Three alternative models of DNA replication
DNA REPLICATION
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication
(Layer 1)
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication
(Layer 2)
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication
(Layer 3)
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication
(Layer 4)
Figure 16.10 Origins of replication in eukaryotes
Figure 16.11 Incorporation of a nucleotide into a DNA strand
Antiparallel Arrangement of Double Strands
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Figure 16.12 The two strands of DNA are antiparallel
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The carbons of the
deoxyribose sugar are
numbered
#3 carbon attached to
an -OH group
#5 carbon holds the
phosphate molecule
of that nucleotide
#3 ready to bond
with another
nucleotide to form a
polynucleotide link (5’
to3’)
Notice complimentary
strand in opposite
direction (5’ to 3’)
DNA always grows 5’
to 3’ never 3’ to 5’
Definitions

Origins of Replication – where
replication of the DNA molecule
begins
Bacteria – circular DNA – 1 origin of
replication (RF)
 Eukaryotes – multiple origins of
replication (ORFS)

• ORF = Replication Fork
More Definitions
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DNA Polymerases – enzymes that
catalyze DNA replication
Leading Strand – Synthesized
continuously towards the replication fork
by the DNA polymerase in one long
fashion
Lagging Strand – Synthesized by short
fragments away from the replication fork
by the DNA polymerase
Definitions Cont’
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Ligase – combines (joins) short fragments
Primer – starts replication of DNA (in this
case it’s RNA)
Primase – an enzyme that joins the RNA
nucleotides to make the primer
Helicase – an enzyme that untwists the
double helix at the replication fork
Nuclease – a DNA cutting enzyme
DNA REPLICATION -VIDEO
Figure 16.13 Synthesis of leading and lagging strands during DNA replication
Figure 16.14 Priming DNA synthesis with RNA
Figure 16.15 The main proteins of DNA replication and their functions
Figure 16.16 A summary of DNA replication
Figure 16.17 Nucleotide excision repair of DNA damage
A PROBLEM!

The end of the
leading strand was
initiated with an RNA
primer
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Normally removed by
other DNA polymerase
Removal of gaps by
DNA Polymerase
doesn’t work on
lagging strand end
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RNA primer removed
with no replacement
A GAP!
SHORTER AND
SHORTER FRAGMENTS?
Prokaryotes have circular DNA – no problem at
ends (there aren’t ANY!
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Eukaryotes – have special terminal
sequences of 6 nucleotides that repeat
from 100-1000 times with no genes
included
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Telomers
Protect more internal gene materials from
being eroded
Germ cells / sex cells have a special
enzyme (telomerase) that actually restore
shortened telomers
Somatic cells – telomer continues to
shorten and may play a role in aged cell
death
Cancer cells

A telomerase prevents very short lengths
Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes
Ribonucleic Acid (RNA)

Structure of RNA

Nucleotide monomer
• Phosphate
• Pentose sugar = ribose (extra oxygen)
• Nitrogenous base (A/G/C/U)
• Single stranded
• 3 types (mRNA, tRNA, rRNA)
Synthesis of RNA transcription
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DNA acts as a template, but only one strand
of DNA utilized at a given time
This exposed strand is controlled by specific
enzymes that pair the DNA nucleotides with
free RNA nucleotides which are also present
in the nucleus
These RNA nucleotides form a single
stranded RNA nucleic acid
DNA = ATTGGCT
RNA = UAACCGA
Short segments of DNA are transcribed at a
time with start and stop messages
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic
information (Layer 1)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic
information (Layer 2)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic
information (Layer 3)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic
information (Layer 4)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic
information (Layer 5)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer
1)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer
2)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer
3)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer
4)
Figure 17.6 The stages of transcription: elongation
Three types of RNA
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mRNA : messenger RNA
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tRNA : transfer RNA
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Transcribed from a specific segment of DNA
which represents a specific gene or genetic
unit
Transcribed from different segments of DNA
and their function is to find a specific amino
acid in the cytoplasm and bring it to the
mRNA
rRNA : ribosomal RNA
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Transcribed at the nucleolus - with proteins
function as the site of protein synthesis
Three types of RNA
Protein Synthesis = Translation
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Ribosomes = sites of protein synthesis
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30% - 40% protein
60% - 70% RNA (rRNA)
Assembled in nucleus and exported via nuclear pores
Antibiotics can paralyze bacterial ribosomes, but not
eukaryotic ribosomes (not targeting them)
2 ribosomal subunits –a large and a small
Small subunit has been used as a means of classifying
different bacteria and different invertebrates (16S)
• Eukaryotes – 18S
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There are three sites on the ribosome that are involved
in protein synthesis
Ribosomes bring mRNA together with amino
acid bearing tRNA’s
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Three ribosomal sites
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P Site (peptidyl-tRNA) holds the tRNA carrying
the growing peptide chain after several amino
acids have been added
A site (aminoaccyl-tRNA) holds the next single
amino acid to be added to the chain
E site (exit site) site where discharged tRNA
minus amino acids leave ribosome
Figure 17.15 Translation – the basic concept
Preparation of Eukaryotic mRNA
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RNA splicing- a cut and paste job to
remove nucleotides from transcribed
mRNA
8000 nucleotides transcribed but the
average gene contains 1200+
nucleotides
 Long non-coding segments (introns)
interspersed between coding segments
(exons) expressed via amino acids
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Figure 17.17 The initiation of translation
Figure 17.18 The elongation cycle of translation
Protein Synthesis (cont’)
Initiation – elongation - termination
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Starting at one end of the mRNA, the small
ribosomal subunit associates with the mRNA and
accepts the first tRNA with its activated amino
acid attached = Initiation
tRNA associate with a triplet codon exposed on
the mRNA – these are 3 nitrogenous bases that
bond with 3 complementary bases exposed
(anticodon) on the tRNA opposite the attached
amino acid
Wobble
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Aren’t 61 tRNAs, are 54tRNAs
Figure 17.4 The dictionary of the genetic code
Figure 17.3 The triplet code
tRNA complexes with its amino acid in the
cytoplasm using ATP – activated tRNA
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The activated tRNA-amino acid complex
moves towards the ribosomal area and
find a triplet codon exposed that is
complementary to the anticodon of the
tRNA
The first activated tRNA-amino acid, after
its anticodon is bound to the mRNA
codon, associates with the large
ribosomal subunit which now joins the
smaller subunit and the mRNA and the
tRNA
(TAKE A BREATH!)
The first tRNA and its amino acid now
occupy the P site of the large ribosomal
subunit
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Review – at this point the 2 part ribosome
is assembled, the mRNA has started to be
read, and one tRNA plus amino acid is
occupying the P site
That means the adjacent A site is free to
accept a second activated tRNA and its
amino acid, but only if the anticodon of
this tRNA matches the next three base
pairs exposed (codon)
Protein Synthesis continued
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At this point, there are 2 tRNA-amino acid
complexes adjacent to each other –
Elongation involved one amino acid being
added in a three step process:
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Codon recognition – the mRNA codon in the A
site matches with the anticodon of the tRNA –
amino acid complex
Peptide bond formation between the new
amino acid in the A site and the amino acid
(later peptide) in the P site
Translocation
Translocation – the ribosome moves the tRNA into
the A site, and its attached peptide to the P site, as
the previous tRNA from the P site moves to the E
(Exit) site and leaves the ribosome
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Review: once this process is under way,
an activated tRNA with its amino acid
finds an exposed codon in the A site,
attaches via H-bonds, then forms a
peptide bond with the polypeptide
associated with the tRNA sitting in the
adjacent P site. For a moment, the
longer polypeptide chain is only attached
to the tRNA in the A site. Now the entire
ribosome shifts so that the………
Yet More Protein Synthesis
The empty tRNA from the P site
moves in to the E site and leaves
the ribosome
 As the tRNA with the polypeptide
chain moves from the A site to the
now empty P site ….exposing a new
codon.
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GUESS WHAT HAPPENS NEXT?!
A question?
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Every time a new codon is exposed in the
A site, a specific tRNA-AA complex moves
into the site. What originally terermined
this mRNA Codon
The Answer!
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The original DNA that was transcribed
This elongation of 1 AA takes about 0.1 s
Termination – the above continues
(dozens to hundreds or more AA added)
until the STOP CODON is reached (codon
at the end of the mRNA)
This codon does not have a matching
tRNA anticodon so the tRNA-AA attaches
in the A site and the tRNA moves to the E
site and releases the polypeptide chain
FIANALLY - SUMMARY
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The take home message:
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At the ribosome, the genetic language
of DNA is translated into a different
language – Via RNA – into the
functioning language of PROTEINS!!!!
Figure 17.17 The initiation of translation
Figure 17.18 The elongation cycle of translation
Figure 17.19 The termination of translation
Figure 17.20 Polyribosomes
Table 17.1 Types of RNA in a Eukaryotic Cell
Figure 17.23 The molecular basis of sickle-cell disease: a point mutation
Figure 17.24 Categories and consequences of point mutations: Base-pair insertion
or deletion
Figure 17.24 Categories and consequences of point mutations: Base-pair
substitution
Figure 17.25 A summary of transcription and translation in a eukaryotic cell
Figure 18.19 Regulation of a metabolic pathway
Control of Protein Synthesis
Regulation of Gene Expression
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Every cell has the same numbers and
types of chromosomes
Development and normal gene function
requires precise gene expression in an on
and off manner
Operon – cluster of gene segments on
DNA and its controlling segments
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Repressible
Inducible
Regions of the Operon (DNA)
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Promoter region : promotes transcription
by binding with RNA polymerase
Operator region : binds a regulatory
protein or chemical
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Overlaps with the RNA polymerase binding
site
Structural genes : code for a particular
peptide or several peptides
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Start or stop codes
Figure 18.20a The trp operon: REPRESSIBLE
Figure 18.20b The trp operon: regulated synthesis of repressible enzymes (Layer 1)
Figure 18.21a The lac operon: INDUCIBLE
Figure 18.21b
The lac operon: regulated synthesis of inducible enzymes
Figure 19.7 Opportunities for the control of gene expression in eukaryotic cells