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Introduction to Cells &
Microscopy
Introduction to Cells & Microscopy
5.1 What Features Make Cells the Fundamental Units of Life?
5.1 What Features Make Cells the Fundamental Units of Life?
Microscopes:
Magnification: Increases apparent size
Resolution: Clarity of magnified object –
minimum distance two objects can be apart
and still be seen as two objects.
Figure 5.3 Looking at Cells (Part 1)
Two basic types of microscopes:
Light microscopes—use glass lenses and
light. Resolution = 0.2 µm
Electron microscopes—electromagnets focus
an electron beam. Resolution = 0.2 nm
Figure 5.3 Looking at Cells (Part 2)
Figure 5.3 Looking at Cells (Part 3)
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5.1 What Features Make Cells the Fundamental Units of Life?
5.1 What Features Make Cells the Fundamental Units of Life?
The plasma membrane:
PARTS OF CELLS:
•  Is a selectively permeable barrier
ALL CELLS
•  Allows cells to maintain a constant internal environment
The plasma membrane is the outer
surface of every cell.
•  Is important in communication and receiving signals
The cytoplasm the inner contents of
every cell.
The cytoplasm:
•  Often has proteins for binding and adhering to adjacent cells
•  Is a concentrated aqueous environment (cytosol)
•  It is very crowded with high concentrations of molecules and
proteins, some as a colloidal suspension
Figure 5.4 A Prokaryotic Cell
5.1 What Features Make Cells the Fundamental Units of Life?
Two types of cells: Prokaryotic and
eukaryotic
Bacteria and Archaea are prokaryotes.
The first cells were probably prokaryotic.
Eukarya are eukaryotes—the DNA is in a
membrane-enclosed compartment called the
nucleus. They also contain organelles such
as mitochondria and chloroplasts. Cells are
larger (>10,000 in volume)
The Gram Stain and the Bacterial Cell Wall
Figure 5.7 Eukaryotic Cells (Part 1)
Outside of cell
Cell wall
(peptidoglycan)
Gram
Positive
Plasma
membrane
5 µm
Inside of cell
Outer membrane
of cell envelope
Outside of cell
Periplasmic space
Gram
Negative
Cell
envelope
Peptidoglycan
layer
Periplasmic space
5 µm
Plasma
membrane
Inside of cell
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Central Dogma of Molecular
Biology
Introduction to Cells & Microscopy
From DNA to Protein: Gene Expression
• Central Dogma: from Genes to Proteins
• Replication of the genes
• Transcribing the information
• Translating the nucleotide sequence into
protein sequence
–  The Genetic Code
–  Protein Biosynthesis
• Control of gene expression
–  prokaryotic
–  eukaryotic
Central Dogma
The central dogma of molecular biology
Replication
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DNA and Replication
Information Flow
DNA and Its Role in Heredity
• Evidence for the Gene and that it is
made of DNA
–  Avery experiments
–  Hershey-Chase experiments
• Structure of DNA
–  Franklin-Wilkins experiments
–  Watson-Crick structure
–  Messelson-Stahl experiments
• DNA Replication
• DNA Repair
13.1 What Is the Evidence that the Gene Is DNA?
Frederick Griffith, determined that a
transforming principle from dead cells
produced a heritable change in the live
cells….. What was this transforming
principle?
Oswald Avery (1944) treated samples to
destroy different molecules; if DNA was
destroyed, the transforming activity was lost.
There was no loss of activity with destruction of
proteins, carbohydrates, or lipids.
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Figure 13.2 Genetic Transformation by DNA (Part 1)
Figure 13.2 Genetic Transformation by DNA (Part 2)
RNAse treated
Figure 13.3 Bacteriophage T2: Reproduction Cycle
Protease treated
DNAse treated
Figure 13.4 The Hershey–Chase Experiment (Part 1)
Hershey-Chase experiment:
•  Used bacteriophage T2
virus to determine whether
DNA, or protein, is the
genetic material
•  Bacteriophage proteins
were labeled with 35S; the
DNA was labeled with 32P
T2 phage (virus) life cycle
Figure 13.4 The Hershey–Chase Experiment (Part 2)
DNA and Its Role in Heredity
• Structure of DNA
– Franklin-Wilkins experiments
– Watson-Crick structure
– Messelson-Stahl experiments
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Figure 13.6 X-Ray Crystallography Helped Reveal the Structure of DNA
13.2 What Is the Structure of DNA?
•  The crucial piece of evidence for DNA structure
came from X-ray “crystallography.” Wilkins learned
how to purify DNA and make regular fiber patterns.
Rosalind Franklin performed the X-ray
diffraction and deduced there was a helix.
•  Francis Crick saw the data at a seminar Wilkins gave
and also deduced there was a helix and the size
parameters.
•  James Watson discovered how the bases went together
(complementarity) using Chargaff rules (A=T, G=C).
Rosalind Franklin
•  Watson & Crick published their structure in 1953.
Beautiful example of how structure predicted
function.
Figure 13.7 DNA Is a Double Helix (Part 1)
Chargaff’s Rule
Photo 13.2 Computer-simulated space-filling model of DNA.
Video: Computer-simulated space-filling model of DNA.
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Figure 13.7 DNA Is a Double Helix (Part 2)
13.2 What Is the Structure of DNA?
Other aspects of DNA structure that
became apparent:
• Genetic material is susceptible to
mutation—a change in information—
possibly a simple alteration to a
sequence.
(34 Å)"
sugar–phosphate
backbone
(phosphodiester
bonds)
Right-handed, antiparallel, doublestranded helix. With the “base
complementarity,” it explains
genetic material:
•  Storage of genetic information
•  Replication
•  Information retrival
Experimental Proof of Structure:
• Access to DNA by proteins is possible
as a sequence-dependent binding
interactions through major groove.
Figure 13.11 The Meselson–Stahl Experiment (Part 1)
Messelson and Stahl Experiment:
Given the double-helix, how is
DNA replicated? Three
possible replication patterns:
§  Semiconservative: Parent serves
as a template and new molecules
have one old and one new strand
§  Conservative: Original helix only
serves as a template
§  Dispersive: Parent fragments
serve as templates, assembling
old and new parts into molecules
They used density labeling to
distinguish parent DNA strands from
new DNA strands. DNA was labeled
with a heavy isotope, 15N, making it
more dense.
Figure 13.11 The Meselson–Stahl Experiment (Part 2)
13.3 How Is DNA Replicated?
• DNA Replication
Arthur Kornberg showed that DNA contains
information for its own replication.
He combined in a test tube: DNA, the four
deoxyribonucleoside triphosphates
(dNTPs–monomers), DNA polymerase,
salts (Mg+2), and buffer.
The DNA served as a template for
synthesis of new DNA.
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Figure 13.12 Each New DNA Strand Grows from Its 5´ End to Its 3´ End (Part 1)
13.3 How Is DNA Replicated?
A large protein complex—the replication complex
—does the work.
Proteins in the Complex:
s  Helicase - unwinds double helix
s  Single-stranded binding protein - maintains ssDNA
s  Primase- makes the primer (small RNA)
s  DNA Polymerase III - does the synthesis
s  DNA Polymerase I - removes primer, fills in gaps
s  DNA ligase - makes final phosphodiester bond
Start point: All chromosomes have a region called
origin of replication (ori). Proteins in the
replication complex bind to a DNA sequence in ori.
Figure 13.14 DNA Polymerase Binds to the Template Strand (Part 2)
The Replicaton Fork
Replication
Fork
Leading strand
3
’
DNA polymerases need a primer.
RNA polymerase (primases) do not.
5
’
Primer
Primase
3’d5’
DNA helicase uses energy from ATP
hydrolysis to unwind the DNA.
Single-strand binding proteins keep
the strands from getting back together.
Lagging strand
Figure 13.20 Telomeres and Telomerase (A)
Problem #2: Synthesis of both strands
What about the last Okazaki fragment at the end of the
chromosome (the telomere)?
Leading strand
No synthesis of other strand!"
Lagging strand
No DNA synthesis occurs because there is no 3’ end to extend
—a single-stranded bit of DNA is left at each end. If left, these
single-stranded regions would be removed after replication;
after many replications, continued chromosome shortening
would lead to cell death.
Telomere"
Leading
Parental Stand"
Lagging
Parental Stand"
3’-end"
Looped around so that polymerase III can synthesize 5’ g 3’
from both strands…and in the same physical direction"
Lagging strand is released
when it gets too big (~2000 bp)"
Okazaki
fragments"
New primer needed. Pieces of lagging
strand called Okazaki fragments"
Replication Fork
Direction"
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Figure 13.20 Telomeres and Telomerase (A)
What about the last Okazaki fragment at the end of the
chromosome (the telomere)?
No DNA synthesis occurs because there is no 3’ end to extend
—a single-stranded bit of DNA is left at each end. If left, these
single-stranded regions would be removed after replication;
after many replications, continued chromosome shortening
would lead to cell death.
Solution:
1) Telomeres– repetative DNA
2) Telomerase– fill in using self-contained primer
to repeated DNA
13.3 How Is DNA Replicated?
Eukaryote chromosomes have repetitive
sequences at the ends called
telomeres.
These repeats are protective and prolong
cell division, especially in rapidlydividing cells, like bone marrow.
Telomerase contains an RNA sequence
—acts as template for telomeric DNA
sequences.
Figure 13.20 Telomeres and Telomerase (B)
DNA and Its Role in Heredity
• DNA Repair
3’d5’
Telomeric DNA filled in ends
Figure 13.21 DNA Repair Mechanisms (A)
13.4 How Are Errors in DNA Repaired?
DNA polymerases make mistakes in
synthesis (1/10,000), plus DNA can be
damaged in living cells.
Cells have three repair mechanisms:
• Proofreading (1/10,000)
• Mismatch repair (1/100)
• Excision repair
correct base
wrong base
Phosphodiester bond made
Phosphodiester bond hydrolyzed
Detected by 3’ à 5’
exonuclease activity (part
of DNA polymerase)
Phosphodiester bond made
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Figure 13.21 DNA Repair Mechanisms (B)
Figure 13.21 DNA Repair Mechanisms (C)
The newly replicated DNA is scanned for mistakes
by other proteins.
A mismatch repair mechanism detects mismatched
bases—the new strand has not yet been modified
(e.g., methylated in prokaryotes) so it can be
recognized.
If mismatch repair fails, the DNA is mutated.
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DNA can be damaged by radiation, chemicals
in the environment, and random
spontaneous chemical reactions.
Excision repair: Enzymes constantly scan
DNA for damaged bases—they are excised
and DNA polymerase I adds the correct
ones.
14.2 How Does Information Flow from Genes to Proteins?
Gene Expression:
Transcription and
Translation
Central Dogma
RNA is key to this process:
•  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 bond formation and provides
structure for the ribosome
Central Dogma
The central dogma of molecular biology
Replication
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)
•  How does genetic information get from the nucleus to the cytoplasm?
Francis Crick proposed TWO hypotheses, based on
their structure of DNA:
•  Messenger hypothesis—a complementary copy of one DNA strand of
the gene is made. The “transcript” travels from nucleus to cytoplasm
carrying information as codons (packages of information encoding the
protein).
•  Adapter hypothesis—an adapter molecule exists in the cell that can
bind amino acids, and recognize a nucleotide sequence, or these
“codons.” These adapter molecules must contain anticodons
complementary to these codons, their recognition based on the
complementary base pairing found in the DNA. During protein
biosynthesis, these adaptor molecules carry amino acids in proper
sequence to interpret or decypher the sequence of the polypeptide
chain—translation.
•  What is the relationship between a DNA sequence and an amino acid
sequence?
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Central Dogma: in Eukaryotes
Prokaryotes and eukaryotes differ in execution of the
Central Dogma
Transcription
Transcription components:
•  A DNA template for base pairings—one
of the two strands of DNA
•  Nucleoside triphosphates
(ATP,GTP,CTP,UTP) as substrates
•  An RNA polymerase enzyme
Transcription: Initiation
Transcription
Transcription occurs in three phases:
•  Initiation
•  Elongation
•  Termination
Transcription: Initiation
Initiation requires a promoter—a
special sequence of DNA.
RNA polymerase binds to the promoter.
Promoter tells RNA polymerase where to
start and which strand of DNA to
transcribe.
Part of each promoter is the initiation
site.
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Transcription: Elongation
Transcription: Elongation
Elongation: RNA polymerase unwinds DNA
about ten base pairs at a time; reads
template in 3’ to 5’ direction, synthesizes
RNA in the 5’ to 3’ direction.
The RNA transcript is antiparallel to the DNA
template strand, and adds nucleotides to its
3’ end.
NTPs incorporate NMP and PPi is a product!
RNA polymerases do not proofread and correct
mistakes (so only initial error rate of 1/10,000).
Transcription: Termination
Transcription: Termination
Termination: Is specified by a specific
DNA base sequence.
Mechanisms of termination are complex
and varied.
For some genes the transcript falls away
from the DNA template and RNA
polymerase—for others a helper
protein pulls it away.
Gene Structure
Gene Structure
In prokaryotes transcription and translation are done
at the same place and time.
In eukaryotes a nucleus separates transcription and
translation.
In prokaryotes, many genes are organized in polycistrons.
Geneall
A genes are
Gene
B
Gene CAlso, they
In
eukaryotes,
monocistronic.
may
promoter
terminator
have noncoding sequences, called introns, with the coding
sequences called exons.
Initiation of transcription"
5’-flanking
End of transcription"
3’-flanking
Cistrons all have
one promoter toGene
which RNA polymerase
binds (with the
promoter
terminator
help of other molecules),
andexon
at theexon
other end
exon
exonof the gene, a terminator
intron
intron polymerase
intron
sequence which signals
where RNA
should end
transcription.
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Post-transcriptional Modification
Post-transcriptional Modification
RNA splicing removes introns and splices exons together.
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.
Consensus sequences are short sequences between exons and introns.
snRNPs binds here, and also near the 3′ end of the intron.
In the nucleus, pre-mRNA is modified in TWO ways at
each end:
1)  The 5’ end is “capped.” G cap (modified guanosine
triphosphate) is added. The cap protects mRNA from being
digested by ribonucleases and facilitates binding to ribosome
Stop Codon"
during translation.
Start Codon"
3’-Untranslated Region (3’-UTR)"
5’-Untranslated Region (5’-UTR)"
Exon 1
intron
Exon 2
Exon 1
Exon 2
intron
Exon 3
Exon 3
Open Reading Frame (ORF)"
Translation: The Genetic Code
(Open Reading "
Frame (ORF)"
20-21 Pol-A
"
site"
bases"
{
Newly transcribed pre-mRNA is bound at ends by snRNPs—small
nuclear ribonucleoprotein particles.
Pol-A signal"
5’-UTR"
3’-UTR"
2)  The 3’ end is poly-adenylated. A Poly-A tail is added 20-21
bases after the poly-A “signal” (AAUAAA) sequence, which is
after last codon. This sequence signals a nuclease to cut the
pre-mRNA; then another enzyme adds 100 to 300 adenines.
May assist in export from nucleus; important for stability of
mRNA.
Translation: The Genetic Code
The genetic code is redundant.
The genetic code is universal.
The genetic code: Specifies which amino
acids will be used to build a protein
Codon: A sequence of three bases—each
codon specifies a particular amino acid.
Start codon: AUG—initiation signal for
translation.
Stop codons: UAA, UAG, UGA—stop
translation and polypeptide is released.
Translation: tRNA
Translation: tRNA
tRNA, the adapter molecule, links
information in mRNA codons with
specific amino acids.
For each amino acid, there is a
specific type or “species” of tRNA.
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Translation: tRNA
tRNAs must deliver amino acids
corresponding to each codon
The conformation (three-dimensional shape) of
tRNA results from base pairing (hydrogen
bonding) within the molecule.
3‘-end is the amino-acid attachment site—binds
covalently.
At the other end (middle of the tRNA sequence) is
the Anticodon—site of base pairing with mRNA.
Unique for each species of tRNA.
Translation: tRNA
Example:
5’-CGG-3’ Coding strand (Crick; sense)
DNA codon for arginine: 3’-GCC-5’ Template strand (Watson; antisense)
Complementary mRNA: 5’-CGG-3’
Has the actual Codons
Anticodon on the tRNA: 3’-GCC-5‘
This tRNA is charged with arginine. Antisense to the Codons
For some tRNAs, there are multiple codons; e.g., that for
alanine, GCA, GCG, GCC, and GCU. These are
recognized by the same tRNA. This is possible due
to Wobble: lack of specificity for the base at the
3‘-end of the codon (5‘–end of the anticodon in
the tRNA).
Wobble base
Translation: tRNA
Translation: Protein Biosynthesis
Aminoacyl-tRNA synthetases—charge
tRNA with the correct amino acids.
Each enzyme is highly specific for one
amino acid and its corresponding
tRNA; the process of tRNA charging is
called the second genetic code.
The enzymes have three-part active
sites: They bind a specific amino acid,
a specific tRNA, and ATP.
Translation: Protein Biosynthesis
Ribosome: the workbench—holds
mRNA and charged tRNAs in the
correct positions to allow assembly of
polypeptide chain.
Ribosomes are not specific, they can
make any type of protein.
Translation: Protein Biosynthesis: Ribosome Structure
Ribosomes have two subunits, large and small. When not active in
translation, the subunits exist separately.
•  The small subunit (40S) has one ribosomal RNA (rRNA) (18S) and 33
proteins.
•  The large subunit (60S) has three molecules of rRNA (28S, 5.8S, 5S)
and 49 different proteins.
•  Ribosomal subunits are held together by ionic and hydrophobic forces
(not covalent bonds) (80S).
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Translation: Protein Biosynthesis
Translation: Protein Biosynthesis; Initiation
Like transcription, translation also
occurs in three steps:
•  Initiation
•  Elongation
•  Termination
Translation: Protein Biosynthesis; Initiation
Initiation:
An initiation complex forms—a charged
tRNA and small ribosomal subunit, both
bound to mRNA: at the correct spot (AUG)
•  In prokaryotes, rRNA binds to mRNA
recognition site “upstream” from start codon
(Shine-Delgarno sequence).
•  In eukaryotes, the small subunit binds to the 5‘G-cap on the mRNA and moves until it reaches
the start codon (Kozak rule).
Translation: Protein Biosynthesis; Elongation
Elongation: The second charged tRNA
enters the A site.
Large subunit catalyzes the two-part
peptidyltransferase reaction:
Ternary complex
–  It breaks bond between tRNA in P-site
and its amino acid.
–  Peptide bond forms between that amino
acid’s (or later that peptide) carboxyl and
the amino acid on tRNA in the A-site
(amino).
IF2
GTP
The rRNA has this activitiy…..catalytic RNA
Translation: Protein Biosynthesis; Elongation
Translation: Protein Biosynthesis; Elongation
EF-Tu
Translocation
(GTP hydrolysis)
GTP
Decoding
(GTP hydrolysis)
Peptidyltransferase
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Translation: Protein Biosynthesis; Elongation
Translation: Protein Biosynthesis; Elongation – REVIEW
When the first tRNA has released its
methionine, as well as subsequent tRNAs, it
moves to the E site and dissociates from the
ribosome—can then become charged again.
Elongation has these steps repeated (decoding,
peptidyltransferase, translocation), assisted by
proteins called elongation factors:
–  EF-Tu/EF-Ts for decoding
–  EF-G for translocation
These steps require energy (2GTP).
© One at decoding in the A-site.
© One at translocation.
Translation: Protein Biosynthesis; Termination
Translation: Protein Biosynthesis; Termination
Termination: Translation ends when a
stop codon enters the A site.
Stop codon binds a protein release
factor (termination factor)—allows
hydrolysis of bond between
polypeptide chain and tRNA on the P
site.
Polypeptide chain separates from the
ribosome—C terminus is the last
amino acid added.
Translation: Protein Biosynthesis; Termination
Translation: Protein Biosynthesis; Termination
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Translation: Protein Biosynthesis
Post-translational Events
Post-translational Events
Post-translational aspects of protein synthesis:
1) FOLDING: Polypeptide emerges from the
ribosome and folds into its 3-D shape.
2) LOCATION: It may contain a signal sequence
indicating where in the cell it belongs; ER,
Nucleus, lysosome, etc.
3) PROCESSING: post-translational changes in
covalent bonds
Post-translational Events
Post-translational Events
Protein modifications:
•  Proteolysis: Cutting of a long
polypeptide chain into final products, by
proteases
•  Glycosylation: Addition of sugars to
form glycoproteins, by
glycosyltransferases
•  Phosphorylation: Addition of phosphate
groups catalyzed by protein kinases—
charged phosphate groups change the
conformation
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Post-translational Events
Control of Gene Expression: Prokaryotes
Example: The lac operon – an
inducible system (polycistronic)
RNA polymerase
Repressor gene promoter operator
Operon genes
Degradation of lactose is a catabolic pathway: genes needed are
induced—turned on when substrate is available.
Control of Gene Expression: Prokaryotes
Control of Gene Expression: Prokaryotes
Start of Transcription
The lac operon is an inducible system.
Other E. coli systems are repressible—
repressed when small molecules called corepressors bind to their repressors.
Binding of co-repressor (effector) to
repressorgrepressor changes shapegbinds
to operatorginhibits transcription
If environment changes and effector is used upgconc.
dropsgco-repressor dissociates from
repressorgrepressor changes shapegdissociates
from operatorg transcription starts (replenishes
effector)
Control of Gene Expression: Prokaryotes
Control of Gene Expression: Eukaryotes?
SUMMARY:
Inducers don’t bind DNA when effector binds-ON
Repressors bind DNA when effector binds-OFF
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Control of Gene Expression: Eukaryotes
Eukaryotic gene regulation can occur at
multiple points in transcription and
translation:
•  Inititation of transcription
w Chromatin remodeling (1)
w Activation of transcriptional initiation (2)
• 
• 
• 
• 
• 
• 
mRNA processing (3)
mRNA transport (4)
mRNA stability (5)
Initiation of translation (6)
Post-translational controls (7)
Protein stability (8)
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