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DNA, RNA, and Protein
Synthesis
Molecular structure of DNA
 Chromosomes contain both nucleic acid and
protein.
 There are two types of nucleic acid, DNA and
RNA.
 Nucleic acid is a polymer of nucleotides, each
comprised of a ribose sugar, a phosphate
group and a nitrogen containing base.
 There are two types of bases, Purines and
Pyrimidines.
In the 1920s, Frederick
Griffith performed
experiments with two
strains of the bacterium
Streptococcus
pneumoniae.
A biological assay
implicates DNA as the
primary genetic
molecule.
In 1952, Alfred D. Hershey
and Martha Chase confirmed
DNA is the genetic material
T2 bacteriophage (virus
that attacks bacteria)
consists of DNA core
packed in protein coat
Phage DNA alone carries
genetic information
A DNA nucleotide
Individual nucleotides of
DNA are linked by
phosphodiester bonds.
This sugar-phosphate
chain forms the
“backbone” of the DNA
molecule.
In the 1950s, chemists Rosalind Franklin and Maurice
Wilkins provided key information about DNA structure using
X-ray crystallography
Erwin Chargaff noted that A = T, and G = C
James D. Watson and Francis
Crick established the general
structure of DNA in 1953
X-ray crystallography
convinced them that the
DNA molecule was helical,
with certain dimensions.
Other evidence for two
polynucleotide chains
running antiparallel to each
other
Key features of DNA:
– double-stranded helix
– diameter is uniform
– the twist is right-handed
– antiparallel strands – two
strands run in different
directions
– Semiconservative replication
uses each parent strand as
template for new strand
DNA Replication
The Meselson – Stahl experiment demonstrating
replication is semi-conservative using density
labeling.
DNA replication is carried out by a
highly complex assembly of proteins
DNA Polymerase III
Helicase
Single-strand binding proteins
Primase
 When DNA polymerase III
reaches previous Okazaki
fragment, it is released
 DNA polymerase I replaces
the primer of previous
Okazaki fragment with DNA,
but leaving a small “nick”
 Finally, DNA ligase
catalyzes formation of the
phosphodiester linkage
that joins the two Okazaki
fragments
Denature and Renature of DNA
 DNA double helix is stabilized by numerous
hydrogen bonds between complimentary base
pairs.
 DNA is denatured or melt if exposed to high
temperature or extreme of pH.
 The melting temperature (Tm) is the
temperature at which half of the DNA molecules
in a sample have been denatured.
 Double helices with an access of G:C base
pairs are more stable and have higher Tm than
helices in which A:T base pairs predominate.
Annealing or hybridization is an
essential and powerful tool
 Labeled DNA fragment can be used as a probe to
find its complement, even in a whole genome.
 The gene of interest can be identified and isolate
from DNA library.
 Specific primers can be designed to amplify the
gene of interest in polymerase chain reaction.
 By observing the extent of annealing between
DNA strands in solution, researchers can
determine the degree of similarity between DNA
molecules from different species.
Decoding Genetic Information:
DNA to RNA to Protein
 Gene expression takes place
in two steps:
 Transcription – makes single-
stranded RNA copy of a DNA
segment
 Translation – uses information
encoded in RNA to make a
polypeptide
http://www.anselm.edu/homepage/jpitocch/genbio/transcrtransl.JPG
Transcription
 In normal prokaryotic and
eukaryotic cells,
transcription requires:
 DNA template for
complementary base pairing
 appropriate ribonucleoside
triphosphates (ATP, GTP, CTP,
and UTP) to act as substrates
 RNA polymerase enzyme
http://www.brooklyn.cuny.edu/bc/ahp/BioInfo/graphics/Transcription.01.GIF
 In prokaryotes, translation of mRNA often begins
before transcription is complete
http://www.phschool.com/science/biology_place/biocoach/images/transcription
Figure 14.2 From Gene to Protein
Each eukaryote gene has one promoter to
which RNA polymerase binds, with the
help of other molecules.
At the other end of the gene there is a
terminator sequence to signal end of
transcription.
Transcription
In the nucleus, pre-mRNA is modified at both ends:
G cap is added at the 5′ end (modified guanosine
triphosphate)—facilitates mRNA binding to
ribosome.
G cap protects mRNA from being digested by
ribonucleases.
Poly A tail added at 3′ end.
AAUAAA sequence after last codon is a signal for
an enzyme to cut the pre-mRNA; then another
enzyme adds 100 to 300 adenines—the “tail.”
May assist in export from nucleus; important for
stability of mRNA.
Eukaryotic genes may have noncoding
sequences—introns.
The coding sequences are exons.
Introns and exons appear in the primary mRNA
transcript—pre-mRNA; introns are removed
from the final mRNA.
Introns interrupt, but do not scramble, the DNA
sequence that encodes a polypeptide.
Sometimes, the separated exons code for
different domains (functional regions) of the
protein.
Intron Splicing
Alternative Splicing
Decoding Genetic Information:
DNA to RNA to Protein
 Francis Crick’s central dogma stated that
information flow is: DNA codes for RNA, and
RNA codes for protein
Replication
Transcription
Translation
Exception to the central dogma:
Viruses: Non-cellular particles that reproduce inside
cells; many have RNA instead of DNA.
Viruses can replicate by transcribing from RNA to RNA,
and then making multiple copies by transcription.
Other viruses such as HIV are
retroviruses.
After infecting a host cell a copy of
the viral genome is incorporated
into the host’s genome to make
more RNA.
Synthesis of DNA from RNA is
reverse transcription.
http://www.brown.edu/Courses/Bio_160/Projects1999/hiv/images/Virion2.jpg
HIV, a retrovirus
Translation
Concurrent transcription and translation in
prokaryotes.
Translation
Transcription and translation are spatially
separated in eukaryotes.
RNA (ribonucleic acid) differs from DNA:
 Usually one polynucleotide strand
 The sugar is ribose
 Contains uracil (U) instead of thymine (T)
Bases in RNA can pair with a single strand of
DNA, except that adenine pairs with uracil
instead of thymine.
Single-strand RNA can fold into complex shapes
by internal base pairing.
Three kinds of RNA in protein synthesis:
 Messenger RNA (mRNA)—carries copy of a
DNA sequence to site of protein synthesis at the
ribosome
 Transfer RNA (tRNA)—carries amino acids for
polypeptide assembly
 Ribosomal RNA (rRNA)—catalyzes peptide
bonds and provides structure
Messenger RNA (mRNA)
 Produced by transcription as a
complementary copy of DNA
 moves from nucleus of
eukaryotic cells into cytoplasm
 serves as template for protein
synthesis
tRNA – the “adapter” molecule
 required to assure specificity in translation of mRNA into
proteins
 tRNAs must read mRNA correctly
 Specific tRNAs must carry correct amino acids
 tRNAs are “adapters”
 link a specific codon with
specific amino acid
 tRNA has three functions:
 carries an amino acid
 associates with mRNA
molecules
 interacts with ribosomes
 Amino acid binding site at 3’ end
of every tRNA (for covalently binding
a specific amino acid)
 Anticodon



On other side of the molecule
three bases which are complementary
to appropriate codon
The codon and anticodon unite by
complementary base pairing.
http://www.wiley.com/legacy/college/boyer/0470003790/structure/tRNA/trna_diagram.gif
 Wobble
 Specificity for base at codon’s third
position is not always observed

Ex: codons for alanine — GCA, GCC,
and GCU — are recognized by the same
tRNA
 Allows cells to produce fewer tRNA
species; but not in all cases — the
genetic code remains unambiguous
http://www.wiley.com/legacy/college/boyer/0470003790/structure/tRNA/trna_diagram.gif
 Aminoacyl-tRNA synthetases
 Attaches amino acids to tRNAs
 Their three-part active sites bind:



a specific amino acid
ATP
a specific tRNA, charged with a high-energy bond
 High-energy bond provides energy for making a peptide
bond
 Translation also occurs in three steps:
 Initiation
 Elongation
 Termination
http://srv2.lycoming.edu/~newman/courses/bio43704/ribosome/ribosome1.jpg
 Initiation
 Start codon (AUG)
designates first amino acid
(methionine) in all proteins
 Large subunit then joins
complex with tRNA at P-site,
all guided by initiation
factors
 Initiation
 The mRNA binds to the small
ribosomal subunit.
 The Shine-Dalgarno
sequence near the 5’ end of
the mRNA base pairs with a
sequence near the 3’ end of
the rRNA of the small
ribosomal subunit.
Elongation: The second charged tRNA enters the
A site.
Large subunit catalyzes two reactions:
 It breaks bond between tRNA in P site and its
amino acid
 Peptide bond forms between that amino acid and
the amino acid on tRNA in the A site
 When the first tRNA has released its methionine,
it moves to the E site and dissociates from the
ribosome—can then become charged again.
 Elongation occurs as the steps are repeated,
assisted by proteins called elongation factors.
 Termination
 When stop codon is
reached…
 Release factor and a
water molecule enter A
site, instead of an
amino acid
 Newly completed
protein separates from
ribosome, and
subunits separate
Protein modifications:
 Proteolysis: Cutting of a long polypeptide
chain into final products, by proteases
 Glycosylation: Addition of sugars to form
glycoproteins
 Phosphorylation: Addition of phosphate
groups catalyzed by protein kinases— charged
phosphate groups change the conformation
High accuracy of protein synthesis
Mutation Results in Mutant Proteins
The genetic disease, sickle-cell anemia, caused by
mutant proteins with single amino acid replacement.
A single amino acid change in such a large complex
hemoglobin could produce profound changes in
activity.
Control of Gene Expression
 Cells conserve energy and resources by making
proteins only when needed.
 Intricate molecular mechanisms must exist in
cells to control the numbers of their many
proteins.
 There are three ways that might achieve this
differential synthesis: transcriptional control,
translational control, and post-transcriptional
control.
Control of Gene Expression
 Cells regulate protein synthesis at any of four
points
 Controlling the rate of mRNA synthesis
 The stability of mRNAs
 Changing the rate of protein synthesis
 The stability of proteins
Control of Gene Expression
 Cells regulate protein synthesis by several
methods:
 Block transcription of the gene
 Hydrolyze the mRNA after it is made
 Prevent translation of mRNA at the ribosome
 Hydrolyze the protein after it is made
 Inhibit the function of the protein
Regulation of mRNA Transcription
 Positive regulation


Gene is not normally transcribed (usually “off”)
Activator protein binds to stimulate transcription
Regulation of mRNA Transcription
One of two types of regulatory protein binds to promoter :
 Negative regulation


Gene is normally transcribed (usually “on”)
Binding of a repressor protein prevents
transcription
Regulation of mRNA Transcription
 E. coli prefers glucose as an energy source, but can
use lactose if glucose is low.
 3 enzymes are required for lactose metabolism
 presence of lactose stimulates production of these
enzymes
 lactose is an inducer
E. coli
http://spacebio.net/modules
Regulation of mRNA Transcription
• enzymes that are
produced in the
presence of an inducer
are said to be inducible
E. coli
• enzymes that are made
all the time are said to
be constitutive
http://spacebio.net/modules
Regulation of mRNA Transcription
 Operon is the whole unit
 promoter, operator, and one or more structural
genes
 Operon containing genes for lactose metabolism:
lac operon
Regulation of mRNA Transcription
 Structural genes specify primary protein structure
— the amino acid sequence
 The three structural genes for lactose enzymes are
adjacent on chromosome
 They share a promoter, and are transcribed together
Regulation of mRNA Transcription
 Prokaryotes shut down transcription by placing
obstacle between promoter and structural
gene
 a protein called a repressor bind to operator—
blocks transcription of mRNA
Regulation of lac Operon
Regulation of lac Operon
 A repressor protein is coded by a regulatory gene.
 The regulatory gene that codes for the lac repressor is
the i (inducibility) gene
 i gene is near lac structural genes, but not all regulatory
genes are near their operons
Regulation of lac Operon
 Regulatory genes like i have their own promoter, called pi
 The i gene is expressed constitutively (expression is
constant).
Regulation of lac Operon
 The repressor protein has two binding sites
 one for the operator
 one for inducer (lactose)
Operator binding
site
Regulation of lac Operon
 Binding inducer to repressor, changes repressor
shape  allows promoter to bind RNA polymerase
 When lactose concentration drops, inducers separate
from repressors — repressor again binds operator,
transcriptions stops
• Promoter is the specific region of the DNA that bound to RNA
polymerase for efficient transcription.
• Two highly conserved, separate nucleotide blocks make up
the promoter of the E. coli lac operon.
• These blocked are designated -10 and -35 upstream of the
mRNA start site +1.
•Mutations in these regions have mild and severe effects on
transcription.
 The sequences required for RNA polymerase
binding and initiation are contained within less
than 40 nucleotides.
 The initial step is the recognition and binding of an
RNA polymerase to the -35 region.
 Subsequently, the -10 region melts into its
component single strands allowing transcription to
begin at +1 position.
 Bacterial RNA polymerase core complexes are
bound by a single initiation factor σ (sigma).
 E coli can control gene expression by activation of
σ factor in response to stimuli.
 σ
70
is the most common initiation factor in E. coli
genome that binds to many other promoters besides lac
promoter.
 σ 32 is activated in response to “heat shock” and directs
polymerase to transcribe specific genes to respond to
this stress.
 σ 54 is activated when cells are starved for nitrogen,
turning on genes that are responsible for uptake of
nitrogen.
Sigma factors produced by E. coli
 Genes are activated when
transcription factors bind specific
DNA sequences near promoter
 If both glucose and lactose are
present, E. coli will first use
glucose.
 Presence of glucose lowers
concentration of cAMP
 thus less CRP binding to
promoter, resulting in less
efficient transcription of
lactose-metabolizing enzymes
 When glucose is used up,
starvation signals accumulate in
the cell, and trigger an activation
of lac operon.
 lac operon can increase efficiency
of the promoter
 A regulatory protein CRP binds
cAMP
 This complex binds to DNA just
upstream of promoter.
 Allows more efficient binding of
RNA polymerase to promoter
Regulation of trp Operon
 A protein is repressible if
synthesis can be turned off
by a biochemical cue (e.g.,
ample supply of that protein)
 The trp operon controls
synthesis of tryptophan — it is
a repressible system
 Gene is normally “on”,
transcribing mRNA and
synthesizing structural
proteins
Regulation of trp Operon
 The repressor must first
bind with a corepressor,
in this case tryptophan
(when its abundant),
activating repressor
 Active repressor turns
gene “off”, blocking
transcription
lac Operon VS trp Operon
 Inducible systems (lac operon)
 Substrate of a metabolic pathway
(inducer) interacts with a
regulatory protein (repressor) —
repressor cannot bind to
operator —allowing transcription
 Control catabolic pathways
(turned on when substrate,
lactose, is present)
lac Operon VS trp Operon
 Repressible system (trp operon)
 Product of a metabolic pathway
(corepressor) interacts with a
regulatory protein (repressor)
allowing it to bind to operator,
blocking transcription
 Control anabolic pathways (turned on
when product, tryptophan, is not
present).
The Attenuation in trp operon
• Attenuation is a method of regulating
transcription by preventing complete mRNA
synthesis.
– The 5’ end of trp operon mRNA, the leader region (region 1) is rich in
tryptophan codon.
– When tryptophan is available, the translation of this region occurs.
– As this happens, the trp mRNA forms a stem-loop structure between
region 3 and 4, transcription is attenuated.
– When the tryptophan level drops, the attenuation is relieved.
– The ribosome is stalled at the leader sequence, different stem-loop
forms (2 and 3), allows the transcription to continue.