Download The Blueprint of Life, From DNA to Protein

The Blueprint of Life,
From DNA to Protein
Chapter 7
Preview
• How does the genetic information pass on
to the next generation?
• How is the information stored in DNA
being used to make protein?
• How are the protein expression regulated?
The Blueprint of Life
• Characteristics of each cell dictated by
information contained on DNA
– DNA holds master blueprint
• All cell structures and processes directed by DNA
Review of DNA basics
•Composed of deoxyribonucleotides
Covalently bonded in chains
5’ end (phosphate)
3’ end (hydroxyl)
•Double-stranded
•Strands are anti-parallel
•Strands are complementary
•Base-pairing rules:
A-T Two H bonds
G-C Three H bonds
•Double helix
•Sugar-phosphate backbone
question
If there are 400 cytosines in a DNA molecule that has 1000
base-pairs, how many adenines does the molecule have?
3’
5’
C
AN
NC
NC
NC
NA
N ANANANAN
G
TN
T TNTNTNTN
NG
NG
NG
NN
3’
5’
Figure 7.1
DNA Replication
DNA Replication
Orig.
New
• Semi-conservative
New
Orig.
DNA Replication
• Semi-conservative
•Bi-directional
•Second round of replication can
start before first is complete
•DNA polymerase “reads” template, adds proper nucleotide to the 3’ end of the
new chain
• Synthesis is 5’  3’ (note: polymerase reads template 3’  5’)
•DNA polymerases generally corrects errors during replication (“proofreading”)
Error rate = 1/billion nucleotides
•DNA polymerases require a primer (they can only add nucleotides onto an existing chain)
question
If a primer were available that bound to the center of the
template molecule in the diagram below, which way would
DNA polymerase move during DNA synthesis?
question
3’
5’
AGTC TG C CTATC GTGAC TA
TCAGAC G GATAG CAC TGAT
3’
5’
5’
3’
5’
5’
question
3’
5’
AGTC TG C CTATC GTGAC TA
TCAGAC G GATAG CAC TGAT
5’
3’
5’
3’
5’
5’
question
3’
5’
AGTC TG C CTATC GTGAC TA
TCAGAC G GATAG CAC TGAT
5’
3’
5’
3’
5’
5’
question
3’
5’
AGTC TG C CTATC GTGAC TA
TCAGAC G GATAG CAC TGAT
5’
3’
5’
3’
5’
5’
question
3’
5’
AGTC TG C CTATC GTGAC TA
TCAGAC G GATAG CAC TGAT
5’
3’
5’
3’
5’
5’
question
3’
5’
AGTC TG C CTATC GTGAC TA
TCAGAC G GATAG CAC TGAT
5’
3’
5’
and so on…
3’
5’
5’
DNA Replication
Replication is initiated at a single distinct region (origin of replication = ori)
5’
3’
3’
5’
*Depicts only a small segment of the circular chromosome
DNA Replication
Replication is initiated at a single distinct region (origin of replication = ori)
5’
3’
5’
5’
A short stretch of RNA (complementary to DNA) is synthesized
3’
5’
DNA Replication
Replication is initiated at a single distinct region (origin of replication = ori)
5’
5’
3’
3’
5’
5’
DNA Replication
Replication is initiated at a single distinct region (origin of replication = ori)
5’
5’
3’
3’
5’
5’
The replication fork (details are shown in Figure 7.6, which is optional)
Leading strand - continuous synthesis
Lagging strand - discontinuous synthesis (Okazaki fragments)
DNA ligase
DNA Replication
• Semi-conservative
•Bi-directional
•Second round of replication can
start before first is complete
DNA Replication
Gene expression
DNA to Proteins - General Principles
-M
I
..
-.-.
C
R
O
.-.
B
I
O
--L
-...
O
G
..
---
Y
Morse code: Distinct series of dots and dashes encode the 26 letters of the
alphabet
Letters strung together make words  sentences  stories
ATGCCCGTAGATGGCCCTGAGCGACCGGACCCTGATGCC
met
pro
val asp
gly
pro
glu
arg
pro
asp
pro
DNA: Distinct series (triplets) of the four nucleotides encode the 20 amino acids
Amino acids strung together make proteins (structural and functional)  cells
 organisms
Gene Expression - Overview
Transcription
Coded by DNA:
Protein A
Protein B
Protein C
Protein D
Protein E
Protein F
Protein G
Protein H
Protein I
Translation
RNA transcripts:
Protein molecules
Protein D
Protein D
Protein D
Protein D
Protein D
Protein D Protein D
Protein D
Protein D
Protein D
D
DD DD
DD
D DD D
DD D DD
D
D DD DD
DD
DD D
D
D D DD
D
Gene: functional unit of DNA that contains information to produce a specific product
Gene Expression - Overview
Transcription
Coded by DNA:
Protein A
Protein B
Protein C
Protein D
Protein E
Protein F
Protein G
Protein H
Protein I
rRNA
tRNA
Translation
RNA transcripts:
Three functional types of RNA:
Messenger (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Protein molecules
Review of RNA basics
Characteristics of RNA
•Composed of ribonucleotides (ribose not deoxyribose); uracil replaces thymine
OH
Characteristics of RNA
Characteristics of RNA
•Composed of ribonucleotides (ribose not deoxyribose); uracil replaces thymine
•Single-stranded
•Sequence is “identical” to a stretch of one strand of DNA; complementary to
the other
RNA
Characteristics of RNA
•Composed of ribonucleotides (ribose not deoxyribose); uracil replaces thymine
•Single-stranded
•Sequence is “identical” to a stretch of one strand of DNA; complementary to
the other
RNA
Characteristics of RNA
•Composed of ribonucleotides (ribose not deoxyribose); uracil replaces thymine
•Single-stranded
•Sequence is “identical” to a stretch of one strand of DNA; complementary to
the other
Template strand
Note: always read (and write) a DNA (or RNA) sequence in the 5’ to 3’
direction, or specify otherwise
Bacterial Gene Expression - Transcription
Transcription initiates at a promoter (sequence “theme” recognized by RNA polymerase)
Transcription stops at a terminator
TTGACA3’
3’AACTGT5’
5’
Bacterial Gene Expression - Transcription
Initiation - RNA polymerase binds to promoter (guided by sigma factor)
Elongation - RNA polymerase synthesizes RNA in 5’  3’ (no primer needed)
Termination -
Terms to note:
Monocistronic
Polycistronic
(prokaryotes only)
Upstream
Downstream
Bacterial Gene Expression - Transcription
5’
3’
3’
A T G A T C T G A G T A T G C G C T
T A C T A G A C T C A T A C G C G A
3’
T A C T A G A C T C A T A C G C G A
5’
5’
Bacterial Gene Expression - Transcription
5’
3’
5’
3’
TTGACA3’
3’ -----------5’
5’
A T G A T C T G A G T A T G C G C T
U A C U A G A C U C A U A C G C G U
3’
A U G A U C U G A G U A U G C G C U
T A C T A G A C T C A T A C G C G A
3’
5’
5’
-----------3’
3’ACAGTT5’
5’
Prokaryotic Gene Expression - Transcription
Prokaryotic Gene Expression - Transcription
Bacterial Gene Expression - Translation
AGAAUGCCCAAUGCGUUACGAUGCCC
•Ribosomes “read” mRNA;
facilitate conversion of the
encoded information into proteins
•Message is read in triplets (codons)
Bacterial Gene Expression - Translation
AGAAUGCCCAAUGCGUUACGAUGCCC
•Ribosomes “read” mRNA;
facilitate conversion of the
encoded information into proteins
•Message is read in triplets (codons)
Bacterial Gene Expression - Translation
AGAAUGCCCAAUGCGUUACGAUGCCC
•Ribosomes “read” mRNA;
facilitate conversion of the
encoded information into proteins
•Message is read in triplets (codons)
•Genetic code is degenerate
But where should the ribosome
start “reading”???
Bacterial Gene Expression - Translation
AGAAUGCCCAAUGCGUUACGAUGCCC
•Ribosomes “read” mRNA;
facilitate conversion of the
encoded information into proteins
•Message is read in triplets (codons)
•Genetic code is degenerate
But where should the ribosome
start “reading”???
•Eukaryotes (moncistronic messages
only) - translation begins at first AUG
•Prokaryotes (monocistronic and
polycistronic messages) - translation
begins at first AUG after a ribosomebinding site
Bacterial Gene Expression - Translation
AGAAUGCCCAAUGCGUUACGAUGCCC
Proper reading frame is critical
AUG
Bacterial Gene Expression - Translation
AGAAUGCCCAAUGCGUUACGAUGCCC
Proper reading frame is critical
Bacterial Gene Expression - Translation
tRNAs are the “keys” that decipher the code
•Each tRNA carries a specific amino acid
•Each tRNA has a specific anticodon,
complementary to a codon, that binds mRNA
Bacterial Gene Expression - Translation
E PA
Initiation
Elongation
translocation
elongation factors
5’
Bacterial Gene Expression - Translation
Termination
Bacterial Gene Expression - Translation
Eukaryotic Gene Expression
Prokaryotic Gene Expression
Eukaryotic Gene Expression
Prokaryotic Gene Expression
Eukaryotic Gene Expression
Eukaryotic Gene Expression
Eukaryotic Gene Expression
Eukaryotic Gene Expression
AAAAAAAAAA
AAAAAAAAAA
Regulation of Gene Expression
• Microorganisms regulate its gene
expression to adapt environment change
– Controls metabolic pathways
• Two general mechanism
– Allosteric inhibition of enzymes
– Controlling synthesis of enzymes
» Directed at making only what is required
Prokaryotic Gene Regulation
Transcription
Coded by DNA:
Protein A
Protein B
Protein C
Protein D
Protein E
Protein F
Protein G
Protein H
Protein I
rRNA
tRNA
Translation
RNA transcripts:
Protein molecules
Protein D
Protein D
Protein D
Protein D
Protein D
Protein D Protein D
Protein D
Protein D
Protein D
D
DD DD
DD
D DD D
DD D DD
D
D DD DD
DD
DD D
D
D D DD
D
Prokaryotic Gene Regulation
Constitutive enzymes
Always produced
Inducible enzymes
Genes turned “on” only when needed
Repressible enzymes
Genes turned “off” when not needed
Regulation of Gene Expression
• Mechanisms controlling transcription
– Often controlled by regulatory region near
promoter
• Protein binds to region and acts as “on/off” switch
– Binding protein can act as repressor or activator
» Repressor blocks transcription
» Activator facilitates transcription
Regulation of Gene Expression
• Repressors
– inhibits gene expression and decreases the
synthesis of enzymes
– usually in response to the overabundance of
an end product
– Repressors block the ability of RNA polymerase to bind
and initiate protein synthesis
– Corepressor
– inducer
Regulation of Gene Expression
• Activators
– turns on the transcription of a gene or set of
genes
• Inducer
• Enzymes synthesized in the presence of inducers
are called inducible enzymes
Regulation of Gene Expression
• Operon model of gene expression
– a set of genes that are controlled by
regulatory proteins
– divided into two regions, the control region
and the structural region
• The control region include the operator and the
promoter
– The operator acts as the “on-off” switch
• The structural region includes the structural genes
– This region contains the genes being transcribed
Operon structure
Operator
Gene 1
Gene 2
Gene 3
Promoter
Promoter –
Binding site
for RNA
polymerase
Operator –
binding site for
the repressor
protein for the
regulation of
gene expression
Structural Genes –
DNA sequence for
specific proteins
Prokaryotic Gene Regulation
DNA-binding proteins
(negative control)
repressor binds, blocking transcription
activator binds, facilitating transcription
(positive control)
Activity of activators/repressors can be controlled
Lac operon
-galactosidase
transport
Lactose  glucose + galactose
Lac operon
Turned “on” only when
lactose is present AND
-galactosidase
transport
glucose levels are low
Is glucose present?
If yes, don’t activate
Is lactose present?
If no, repress
Lactose  glucose + galactose
Lac operon
Turned “on” only when
lactose is present AND
glucose levels are low
Negative control repressor is active if
lactose is absent; inactive
if lactose is present
Positive control - CAP only
binds if cAMP is available
(glucose levels are low)
Glucose transport into cell
lowers cAMP levels