Download DNA Replication and Protein Synthesis PowerPoint

DNA! - Chapter 10
Good luck!
Please go directly to your test seats and
grab a scantron from my desk
Let’s review where we find
our genetic coding ……
What holds our genetic coding?
• Chromosomes
✓ Strands of DNA that contain all of
the genes an organism needs to
survive and reproduce
• Genes
✓ Segments of DNA that specify
how to build a protein
• There can be multiple genes
for a trait/protein
✓ Chromosome maps are used
to show the locus (location)
of genes on a chromosome
The E. Coli genome includes
approximately 4,000 genes
Genetic Variation
✓ Phenotypic variation among organisms is due to genotypic variation
(differences in the sequence of their DNA bases)
✓ Differences exist between species and within a species
• Different genes (genomes) → different proteins
• Different versions of the same gene (alleles)
• Differences in gene expression (epigenetics)
DO NOW:
What are the 3 components that
make up a nucleotide structure?
Structure of DNA
DNA stands for: Deoxyribonucleic acid
DNA is located in the nucleus of eukaryotic cells
and cytoplasm in prokaryotic cells
DNA and RNA are polymers composed of nucleotides
▪ Polymers = DNA and RNA
▪ Monomer = nucleotide
Each nucleotide contains a:
– Nitrogenous base
– 5-carbon sugar
– Phosphate group
Nitrogenous base
(A, G, C, or T)
Phosphate
group
Thymine (T)
Sugar
(deoxyribose)
The 4 Nitrogenous bases of DNA:
Thymine (T)
Cytosine (C)
Pyrimidines
Adenine (A)
Guanine (G)
Purines
What is the difference between
pyrimidines and purines?
Nitrogenous bases bond together to make a BASE
PAIR
Adenine → Thymine
How are
they bonded
Cytosine → Guanine
together?
How many
bonds does
each pair
have?
A sugar-phosphate
backbone is formed by
covalent bonding between
the phosphate of one
nucleotide and the sugar
of the next nucleotide
Nitrogenous bases extend
from the sugar-phosphate
backbone
What does Antiparallel mean?
● Two DNA strands run
parallel but in the
opposite alignment
● Allows nucleotides to
make the needed
hydrogen bonds
5’ - 3’
● These numbers identify the carbons on the sugar backbone
● Start to the right of the “o”
● Asymmetry gives a DNA strand “direction”
Count the carbons
Sugar-phosphate backbone
Phosphate group
Nitrogenous base
Sugar
DNA nucleotide
Phosphate
group
Nitrogenous base
(A, G, C, or T)
Thymine (T)
Sugar
(deoxyribose)
DNA nucleotide
DNA polynucleotide
Notice the →
● Different bonds
● Different bases
● 5’ - 3’ & 3’-5’
Sugar → Deoxyribose or Ribose
What is RNA?
●
●
●
●
Ribonucleic Acid, present in all living cells
Single Stranded
Helps with the creation of proteins!
Has 3 of the same nitrogenous bases DNA
has except uracil takes the place of thymine
DNA Replication
DNA Replication
• Cell Division (mitosis)
✓ Cells must copy their chromosomes
(S phase) before they divide so that each
daughter cell will have a copy
Questions before we start…
1. How does that DNA actually replicated?
2. What form is the DNA in during this?
AT
GC
CG
TA
GC
DNA Synthesis
✓ The DNA bases on each strand act
as a template to synthesize a
complementary strand
✓ The process is semiconservative
because each new double-stranded
DNA contains one old strand (template)
and one newly-synthesized
complementary strand
AT
GC
CG
ATTA
GGCC
CG
TA
GC
A
G
C
T
G
AT
GC
CG
TA
GC
T
C
G
A
C
Enzymes involved:
• Topoisomerase
✓ Unwinds (uncoils) the DNA
• Helicase
✓ An enzyme that separates strands of nucleic acids
“Replication Fork”
• Single Stranded Binding Proteins
✓ Prevent DNA that has been opened at the replication fork during DNA
replication from immediately reestablishing double helix conformation
• RNA Primase
✓ an enzyme that creates a short RNA sequence, called a
primer, tells the DNA polymerase where to start replication
• DNA Polymerase (I and III)
✓ Enzyme that catalyzes the covalent bond between the phosphate of one
nucleotide and the deoxyribose (sugar) of the next nucleotide
Polymerase I: replaces segments of
primer with DNA nucleotides
Polymerase III: binds at the end of the
primer and adds new DNA nucleotides
• Ligase
✓ Joins DNA strands back together (acts as a glue)
3’ end has a free deoxyribose
5’ end has a free phosphate
DNA polymerase:
✓ can only build the new strand in
the 5’ to 3’ direction
✓ Thus scans the template strand in
3’ to 5’ direction
DNA Replication Steps…
1. Initiation
2. Elongation
3. Termination
Initiation
• Primase (a type of RNA polymerase) builds an RNA primer
(5-10 ribonucleotides long)
• DNA polymerase attaches onto the 3’ end of the RNA primer
DNA
polymerase
Elongation
• DNA polymerase uses each strand as a template in the 3’ to 5’
direction to build a complementary strand in the 5’ to 3’ direction
• results in a leading strand and a lagging strand
DNA polymerase
Lagging Strand Creates Okazaki Fragments
• Newly synthesized DNA fragments that are formed on the lagging
template strand during DNA replication
Last step...
Termination
Primers are removed and
replaced with new DNA
nucleotides and the backbone is
sealed by DNA ligase
2 new daughter strands
of DNA!
Eukaryotic vs. Prokaryotic
DNA replication
Review of Enzymes involved
and DNA replication:
– DNA polymerase adds nucleotides to a growing chain
– DNA ligase joins small fragments into a continuous chain
– Helicase unwinds the DNA strand
– RNA Primase tells DNA polymerase where to start; “primer”
– Single Stranded Binding Proteins prevent DNA from coiling
back into a double helix during synthesis
Review...
Leading Strand
1. Topisomerase unwinds DNA and then Helicase breaks H-bonds
2. Primase creates a single RNA primer to start the replication
3. Polymerase slides along the leading strand in the 3’ to 5’ direction synthesizing
the matching strand in the 5’ to 3’ direction
4. The RNA primer is degraded by RNase H and replaced with DNA nucleotides by
DNA polymerase, and then DNA ligase connects the fragment at the start of the
new strand to the end of the new strand (in circular chromosomes)
Review...
Lagging Strand
1. Topisomerase unwinds DNA and then Helicase breaks H-bonds
2. Primase creates RNA primers in spaced intervals
3. Polymerase slides along the leading strand in the 3’ to 5’ direction synthesizing
the matching Okazaki fragments in the 5’ to 3’ direction
4. The RNA primers are degraded by RNase H and replaced with DNA nucleotides
by DNA polymerase
5. DNA ligase connects the Okazaki fragments to one another (covalently bonds the
phosphate in one nucleotide to the deoxyribose of the adjacent nucleotide)
Protein Synthesis
DO NOW:
1. Collect your graded papers on the back table
2. Take out both lab sheets from
yesterday and sit with your group
We are going to analyze our gels!
Protein Synthesis
• DNA provides the instructions for how to build proteins
• Each gene dictates how to build a single protein
• The sequence of nucleotides (AGCT) in DNA dictate the order
of amino acids that make up a protein
Nucleotide sequence of this gene
Protein construction requires a conversion of a
nucleotide sequence to an amino acid sequence
Central Dogma
Protein Synthesis
• Protein synthesis occurs in two primary steps
1
● DNA is transcribed into
mRNA (messenger RNA)
● RNA processing occurs
shortly after
✓Cytoplasm of prokaryotes
✓Nucleus of eukaryotes
2
● mRNA is used by ribosome to
build protein by being
assisted by rRNA and tRNA
● Ribosomes (rRNA) attach to
the mRNA and use its sequence
of nucleotides to determine the
order of amino acids in the
protein
Before we start, let's review the 3
types of RNA we will be talking about
mRNA ~ Messenger RNA; takes the code from the DNA and brings
it to the ribosome. It is made during first step called transcription.
rRNA ~ Ribosomal RNA; combines with proteins to make the
structure of the ribosome.
tRNA ~ Transfer RNA; carries an amino acid to the ribosome to be
able to synthesize the protein during translation.
Steps to create a protein!
1. Transcription
a. RNA Processing
2. Translation
*We will be going over the exact details of each step in both of these processes*
Steps of Transcription and Translation
1. Initiation
2. Elongation
3. Termination
*We will be going over the exact details of each step in both of these processes*
Protein Synthesis
1) INITIATION
• Transcription - Initiation
✓ RNA polymerase binds to a
region on DNA known as the
promoter, which signals the
start of a gene (does not need a primer)
✓ Promoters are specific to genes
■ TATA Box
✓ Transcription factors assemble
at the promoter forming a
transcription initiation complex
– activator proteins help stabilize
the complex
(eukaryotes)
• Transcription - Elongation
✓ RNA polymerase unwinds the
DNA and breaks the H-bonds
between the bases of the two
strands, separating them from one
another.
✓ Base pairing occurs between
incoming RNA nucleotides and
the DNA nucleotides of the gene
(template)
• recall RNA uses uracil
instead of thymine
1) INITIATION
AGTCAT
UCAGUA
• Transcription - Elongation
✓ RNA polymerase unwinds
the DNA and breaks the
H-bonds between the bases
of the two strands, separating
them from one another.
5’
✓ Base pairing occurs between
incoming RNA nucleotides
and the DNA nucleotides of
the gene (template)
• recall RNA uses uracil
instead of thymine
3’
+ ATP
✓ RNA polymerase catalyzes bond to5’
form between ribose of 3’ nucleotide
of mRNA and phosphate of incoming
RNA nucleotide
3’
+ ADP
• Transcription - Elongation
• Transcription - Termination
1) INITIATION
✓ A region on DNA known
as the terminator signals
the stop of a gene
✓ RNA polymerase disengages
the mRNA and the DNA
TRANSCRIPTION COMPLETE!
At the end of Transcription you now have an
almost complete mRNA strand...
● Eukaryotic mRNA has interrupting (noncoding)
sequences called introns, separating the coding
regions called exons.
● Although introns are taken out, they help cell function
and enhance gene expression.
RNA Processing = RNA Splicing
✓ Introns are removed (exons together)
✓ different combinations of exons form different mRNA resulting in multiple
proteins from the same gene
✓ Humans have 30,000 genes but are capable of producing 100,000 proteins
Light pink
regions are
the “introns”
What are
those yellow
regions on
the mRNA?
A CAP and TAIL is added to the mRNA
Cap added to 5’ end: single guanine nucleotide
Tail added to 3’ end: Poly-A tail of 50–250 adenines
Why is the CAP and TAIL important?
These structures increase the stability of the
mRNA as it moves through the cytoplasm and also
help it to interact with the components necessary
for protein synthesis.
Exon Intron
Exon
Intron Exon
DNA
Cap
RNA
transcript
with cap
and tail
Transcription
Addition of cap and tail
Introns removed
Tail
Exons spliced together
mRNA
Coding sequence
Nucleus
Cytoplasm
Protein Synthesis Part 2
Transcription
tRNA
synthesis
1
2
mRNA
mRNAmRNA copy of a gene
is synthesized
mRNA is used by ribosome to build protein
✓Cytoplasm of prokaryotes Ribosomes attach to the mRNA and use its
✓Nucleus of eukaryotes
sequence of nucleotides to determine the
order of amino acids in the protein
✓Cytoplasm of prokaryotes & eukaryotes
Translation
Protein Synthesis Part 2
Transcription
• Translation
tRNA
synthesis
✓ Every three mRNA nucleotides (codon) specify an amino acid
mRNA
Translation
Protein Synthesis Part 2
• Translation
✓ tRNA have an anticodon region that specifically binds to its codon
✓ Each tRNA carries a
specific amino acid
Protein Synthesis Part 2
• Translation - Initiation
✓ Start codon signals where the gene
begins (at 5’ end of mRNA)
✓ We have 1 start codon = AUG
5’
AUGGACAUUGAACCG…
start codon
3’
• Translation- Initiation
✓ Start codon signals where the gene begins (at 5’ end of mRNA)
✓ Ribosome binding site upstream from the start codon binds to
the small ribosomal subunit
✓ This complex recruits the large ribosomal subunit to bind
A site
✓ P site = hold tRNA carrying growing polypeptide chain
✓ A site = Holds tRNA carrying next amino acid in chain
✓ E site = Empty tRNA leaves exit site
A site
Translation Animation
http://highered.mheducation.
com/sites/0072507470/student_view0/chapter3/animation__h
ow_translation_works.html
• Translation - Elongation
✓ The ribosome moves in 5’ to 3’ direction “reading” the mRNA and
assembling amino acids into the correct protein
• Translation - Elongation
✓ The ribosome moves in 5’ to 3’ direction “reading” the mRNA and
assembling amino acids into the correct protein
• Translation - Termination
✓ Ribosome disengages from the mRNA
when it encounters a stop codon
✓ There are 3 STOP codons
= UAA, UAG, UGA
Practice Question!
Translate the following mRNA sequence
AGCUACCAUACGCACCCGAGUUCUUCAAGC
Practice Question!
Translate the following mRNA sequence
AGCUACCAUACGCACCCGAGUUCUUCAAGC
Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine
Practice Question!
Translate the following mRNA sequence
AGCUACCAUACGCACCCGAGUUCUUCAAGC
Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine
Ser – Tyr – His – Thr – His – Pro – Ser – Ser – Ser - Ser
Practice Question!
Translate the following mRNA sequence
AGCUACCAUACGCACCCGAGUUCUUCAAGC
Serine – Tyrosine – Histidine – Threonine – Histidine – Proline – Serine – Serine – Serine - Serine
Ser – Tyr – His – Thr – His – Pro – Ser – Ser – Ser - Ser
S – Y –H– T – H – P – S – S – S - S
Protein Synthesis
• Multiple RNA polymerases can
engage a gene at one time
• Multiple ribosomes can engage
a single mRNA at one time
Transcription
DNA
mRNAs
Translation
Protein Synthesis
• Eukaryotes:
transcription occurs
in the nucleus and
translation occurs in
the cytoplasm
• Prokaryotes:
Transcription and
translation occur
simultaneously in
the cytoplasm
Mutations
A mutation is a change in the nucleotide sequence of DNA
Base substitutions: replacement of one nucleotide with another
– Effect depends on whether there is an amino acid change
that alters the function of the protein
Deletions, insertions, inversions:
– Alter the reading frame of the mRNA, so that nucleotides
are grouped into different codons
– Lead to significant changes in amino acid sequence
downstream of mutation
– Cause a nonfunctional polypeptide to be produced
Mutations
Mutations can be …
● Spontaneous: due to errors in DNA replication (aging)
● Induced by mutagens
● High-energy radiation
● Chemicals
Two major types of mutations:
Point Mutations - changes in 1 or a few nucleotides
- Substitution
- Insertion
- Deletion
Chromosomal Mutations - change in chromosome segment
-
Deletion
Duplication
Inversion
Translocation
Point Mutations
1. Deletion - loss of one or more nucleotides from a gene; can be
segment of chromosome
2. Substitution - change of one nucleotide for another
3. Insertion - additional nucleotide or chromosome segment added
into the existing gene
Normal hemoglobin DNA
Mutant hemoglobin DNA
mRNA
mRNA
Normal hemoglobin
Sickle-cell hemoglobin
Glu
Val
Substitution
Normal gene
mRNA
Protein
Met
Lys
Phe
Gly
Ala
Met
Lys
Phe
Ser
Ala
substitution
deletion
Missing
Met
Lys
Leu
Ala
His
Chromosomal Mutations
1. Deletion - loss of one or more nucleotides from a gene; can be
segment of chromosome
2. Duplication- produces an extra copy of a segment on a chromosome
3. Translocation - part of a chromosome breaks off and attaches to
another
4. Inversion - a change in chromosome result in from reattachment of
a chromosome fragment to the original chromosome, but in the
reverse direction
Duplication
Inversion
Practice Questions
1. Why is DNA synthesis said to be “semiconservative”?
2. What role do DNA polymerase, DNA primase (a type of RNA polymerase), helicase,
topoisomerase, RNase H, and ligase play in DNA replication?
3. What is the difference between how the leading strand and lagging strand are copied
during DNA replication? Why do they have to be synthesized differently in this fashion?
4. What would happen if insufficient RNase H were produced by a cell? What if insufficient
ligase were produced by a cell?
5. What are four key differences between DNA polymerase and RNA polymerase? (“they
are difference molecules” doesn’t count as one!)
6. Compare and contrast codons and anticodons?
7. What is alternative splicing? Why is it necessary in eukaryotes?
8. During translation, what amino acid sequence would the following mRNA segment be
converted into: AUGGACAUUGAACCG?
9. How come there are only 20 amino acids when there are 64 different codons?
10. How come prokaryotes can both transcribe and translate a gene at the same time, but
eukaryotes cannot?