Molecular Biology II
Recombinant DNA Technology
•Biotechnology terminology
•Common hosts and experimental organisms
•Transcription and translation
•Prokaryotic gene organization & expression
Terminology
• Molecular biology-The study of biology on a molecular level
including the structure, function, and makeup of biologically
important molecules such as DNA, RNA, and proteins
• Recombinant DNA technology-a set of techniques for
manipulating DNA, including: the identification and cloning of
genes; the study of the expression of cloned genes; and the
production of large quantities of gene product
• Genetic engineering-the process of transferring DNA from one
organism into another that results in a genetic modification
• Biotechnology-production of goods and services using
biological organisms, systems, and processes
• Molecular biotechnology-rDNA technology + biotechnology
Many scientific disciplines contribute to
molecular biotechnology, which generates a
wide range of commercial products
Common host organisms used
in molecular biotechnology
•
•
•
•
•
E. coli
Yeast (Saccharomyces cerevisiae)
Insect cell lines
Plant cell lines
Animal cell lines
Figure 1.13
studies.
Each experimental organism used in cell biology has advantages for certain types of
Molecular Cell Biology, 7th Edition
Lodish et al.
Copyright © 2013 by W. H. Freeman and Company
Figure 1.13 (Continued) Each experimental organism used in cell biology has advantages for
certain types of studies. Listen to the podcast!
Molecular Cell Biology, 7th Edition
Lodish et al.
Copyright © 2013 by W. H. Freeman and Company
Molecular Cell Biology, 7th Edition
Lodish et al.
Copyright © 2013 by W. H. Freeman and Company
Review protein secretion and protein targeting
• Signal peptide sequences
• Consider gram negative vs.
gram positive bacteria
• Consider eukaryotic cells
• In eukaryotic cells, short
peptide sequences (or
other modifications) tell a
protein where to go
• See MCB Chapter 13 & 14Protein sorting animations
http://bcs.whfreeman.com/lodish7e/#800911__811705__
http://bcs.whfreeman.com/lodish7e/#800911__812066__
Central Dogma of Biology
DNA
transcription
RNA
reverse
transcription
DNA
replication
translation
Protein
$$$
Molecular
Biotechnology
Chemical structure of DNA & RNA
Chemical structure of dsDNA
Chemical structure of dsDNA
Prokaryotic gene expression
In prokaryotes, RNA polymerase binds to the 10 and -35 regions of the promoter relative to
the start site of transcription (+1)
promoter
operator
•Eukaryotic gene organization
•Restriction enzymes
•Cloning vectors
Eukaryotic gene organization
enhancers
silencers
Eukaryotic gene organization & RNA processing
Figure 4.14 Structure of the 5’ methylated cap.
Basic Transcriptional Mechanisms and
mRNA Splicing Animations
Basic Molecular Genetic Mechanisms (animations)
• Life Cycle of mRNA
 http://bcs.whfreeman.com/lodish7e/#800911__812036__
• Basic Transcriptional Mechanisms
 http://bcs.whfreeman.com/lodish7e/#800911__812037__
Post-transcriptional Gene Control (animation)
• mRNA Splicing
 http://bcs.whfreeman.com/lodish7e/#800911__812057__
Prokaryotic vs. eukaryotic gene organization
Alternative splicing of eukaryotic 1° RNA transcripts
Eukaryotic gene expression
Life Cycle of mRNA
Basic Molecular Genetic Mechanisms (animation)
• Life Cycle of mRNA
 http://bcs.whfreeman.com/lodish7e/#800911__812036__
Translation
OrR
Stages of Translation Process
Protein synthesis can be conveniently
divided into four stages:
(1) The binding of amino acids to the
tRNAs
(2) Initiation, in which the components
necessary for translation are assembled at
the ribosome
(3) Elongation, in which amino acids
are joined, one at a time, to the
growing
polypeptide chain;
(4) Termination, in which protein synthesis
halts at the termination codon and the
translation components are released from
the ribosome.
The Binding of Amino Acids to Transfer RNAs
• Cell typically possesses from 30 to 50
different tRNAs
•Collectively, these tRNAs are attached to
the 20 different amino acids.
•Each tRNA is specific for a particular kind
of amino acid.
•All tRNAs have the sequence CCA at the 3’
end, and the carboxyl group (COO ) of the
amino acid is attached to the 2’- or 3’hydroxyl group of the adenine nucleotide
at the end of the tRNA
If each tRNA is specific for a particular amino acid but all amino acids
are attached to
the same nucleotide (A) at the 3’ end of a tRNA, how does a tRNA link
up with its
appropriate amino acid?
The key to specificity between an amino acid and its tRNA is a set of
enzymes called
aminoacyl-tRNA synthetases.
A cell has 20 different aminoacyl-tRNA synthetases, one for each of the 20
amino acids.
Each synthetase recognizes a particular amino acid, as well as all the
tRNAs that accept that amino acid.
Recognition of the appropriate amino acid by a synthetase is based on the
different sizes, charges, and R groups of the amino acids.
The tRNAs, however, are all similar in tertiary structure. How does a
synthetase distinguish among tRNAs?
The recognition of tRNAs by a
synthetase depends on the differing
nucleotide
sequences of tRNAs.
Researchers have identified which
nucleotides are important in recognition
by altering different nucleotides in a
particular tRNA and determining
whether the altered tRNA is still
recognized by its synthetase.
The results of these studies revealed that
the anticodon loop, the DHU-loop, and the
acceptor stem are particularly critical for
the identification of most tRNAs
The attachment of a tRNA to its appropriate amino acid (termed tRNA
charging) requires energy, which is supplied by adenosine triphosphate (ATP):
This reaction takes place in two steps:
Errors in tRNA charging are
rare; they occur in only about 1
in 10,000 to 1 in
100,000 reactions.
This fidelity is due to the presence
of proofreading activity in the
synthetases, which detects and
removes incorrectly paired amino
acids from the tRNAs.
The Initiation of Translation
Prokaryotic Initiation System
The second stage in the process of protein synthesis is initiation.
During initiation, all the components necessary for protein synthesis assemble:
(1) mRNA
(2) the small and large subunits of the ribosome
(3) a set of three proteins called initiation factors
(4) initiator tRNA with N-formylmethionine attached (fMet-tRNAfMet )
(5) guanosine triphosphate (GTP). Initiation comprises three major steps.
The Initiation of Translation
Prokaryotic Initiation System
Initiation comprises three major steps.
First, mRNA binds to the small subunit of the ribosome.
Second, initiator tRNA binds to the mRNA through base pairing between the
codon and anticodon.
Third, the large ribosome joins the initiation complex.
The Initiation of Translation
Prokaryotic Initiation System
The Initiation of Translation
Prokaryotic Initiation System
The sequence covered by the ribosome
during initiation is from 30 to 40 nucleotides
long and includes the AUG initiation codon.
Within the ribosome-binding site is the
Shine-Dalgarno consensus sequence,
which is complementary to a sequence of
nucleotides at the 3’ end of 16S rRNA (part
of the
small subunit of the ribosome).
During initiation, the nucleotides in the
ShineDalgarno sequence pair with their
complementary nucleotides in the 16S
rRNA,
allowing the small subunit of the ribosome to
attach to the mRNA and positioning the
ribosome directly over the initiation codon.
The Initiation of Translation
Eukaryotic Initiation System
The small subunit of the eukaryotic ribosome, with the help of initiation
factors, recognizes the cap and binds there;
the small subunit then migrates along (scans) the mRNA until it locates the
first AUG codon.
The identification of the start codon is facilitated by the presence of a
consensus
sequence (called the Kozak sequence) that surrounds the start codon:
Elongation
Elongation requires
(1) the 70S complex just described
(2) tRNAs charged with their amino acids;
(3) several elongation factors (EF-Ts, EF-Tu, and
EF-G);
(4) GTP.
A ribosome has three sites that can
be occupied by tRNAs; the aminoacyl, or
A site, the peptidyl, or P site, and the
exit, or E site
Termination
The Overall Process of Protein Synthesis
RNA–RNA Interactions in Translation
(1) The process of translation is rich in RNA–RNA interactions. For
example, in
bacterial translation, the Shine-Dalgarno consensus sequence at the 5’
end of the mRNA pairs with the 3’ end of the 16S rRNA (Figure 9.6), which
ensures the binding of the ribosome to mRNA
Polyribosomes
In both prokaryotic and eukaryotic cells, mRNA molecules are translated
simultaneously by multiple ribosomes. The resulting structure—an mRNA
with
several ribosomes attached—is called a polyribosome.
Each ribosome successively attaches to the ribosome- binding site at the 5’ end
of the mRNA and moves toward the 3’ end; the polypeptide associated with
each ribosome becomes progressively longer as the ribosome moves along
the mRNA.
A Comparison of Bacterial and
Eukaryotic Translation
1.Initiator Amino Acid
2.Temporal and Spatial Differences of Gene Ex
3.Longevity of mRNA
4.Sizes and Compositions Ribosomal Subunit
5.Initiation of Transcription
6.Elongation and Termination
The Posttranslational Modifications of Proteins
Translation and Antibiotics
Recombinant DNA cloning procedure
Recombinant DNA cloning procedure
MCB Chapter 5 - Molecular Genetic Techniques (animation)
• Plasmid Cloning
 http://bcs.whfreeman.com/lodish7e/#800911__812047__
Restriction enzymes & DNA methylation
Recognition sequences of some REs
Enzyme
EcoRI
BamHI
PstI
Sau3A1
PvuII
HpaI
HaeIII
NotI
Recognition site
G￬A-A-T-T-C
G￬G-A-T-C-C
C-T-G-C-A￬G
￬G-A-T-C
C-A-G￬C-T-G
G-T-T￬A-A-C
G-G￬C-C
G￬C-G-G-C-C-G-C
Type of cut end
5’ P extension
5’ P extension
3’ P extension
5’ P extension
Blunt end
Blunt end
Blunt end
5’ P extension
Restriction Endonuclease (sticky end)
Restriction Endonuclease (blunt end)
Restriction Endonuclease Type II
Restriction Endonuclease (Type IIS)
Neoschizomers
Mapping of restriction enzyme sites
The Production of Recombinant DNA Molecules In Vitro
Cloning vectors and their insert capacities
Vector system
Host cell
Insert capacity (kb)
Plasmid
E. coli
0.1-10
Bacteriophage l
E. coli
10-20
Cosmid
E. coli
35-45
Bacteriophage P1
E. coli
80-100
BAC (bacterial artificial E. coli
chromosome)
50-300
P1 bacteriophagederived AC
E. coli
100-300
YAC
Yeast
100-2,000
Human AC
Cultured human cells
>2,000
Plasmid cloning vectors
Three important features
1. Cloning site
2. Ori-an origin of replication
3. A selectable marker (ampr)
Plasmid
How we insert DNA fragment in plasmid????
pBR322
ori
The plasmid pBR322 is one of the most commonly used E.coli cloning vectors. pBR322 is 4361 bp in
length and contains: (1) the replicon rep responsible for the replication of plasmid (source – plasmid
pMB1); (2) rop gene coding for the Rop protein, which promotes conversion of the unstable RNA I –
RNA II complex to a stable complex and serves to decrease copy number (source – plasmid pMB1); (3)
bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – transposon Tn3); (4)
tet gene, encoding tetracycline resistance protein (source – plasmid pSC101).
pUC18/19
pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids,
2686 bp in length. They are identical except that they contain multiple
cloning sites (MCS) arranged in opposite orientations. pUC18/19 plasmids
contain: (1) the pMB1 replicon rep responsible for the replication of
plasmid (source – plasmid pBR322). The high copy number of pUC
plasmids is a result of the lack of the rop gene and a single point mutation
in rep of pMB1; (2) bla gene, coding for beta-lactamase that confers
resistance to ampicillin (source – plasmid pBR322); (3) region of E.coli
operon lac containing CAP protein binding site, promoter Plac, lac repressor
binding site and 5’-terminal part of the lacZ gene encoding the N-terminal
fragment of beta-galactosidase (source – M13mp18/19). This fragment,
whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa)
complementation with a defective form of beta-galactosidase encoded by
host (mutation lacZDM15). In the presence of IPTG, bacteria synthesize
both fragments of the enzyme and form blue colonies on media with X-Gal.
Insertion of DNA into the MCS located within the lacZ gene (codons 6-7 of
lacZ are replaced by MCS) inactivates the N-terminal fragment of betagalactosidase and abolishes alfa-complementation. Bacteria carrying
recombinant plasmids therefore give rise to white colonies.
pGEM-3Z
Cloning foreign DNA into a plasmid vector
Alkaline phosphatase-removes
5’ phosphate (P) groups of
DNA molecules; BAP is more
stable but less active than CIP
T4 DNA ligase –joins 5’
phosphate (P) groups of DNA
molecules to 3’ hydroxyl (OH)
groups of DNA
Some antibiotics commonly used as selective agents
Antibiotic
Description
Ampicillin (Amp)
Inhibits bacterial cell wall synthesis; inactivated by blactamase, which cleaves the b-lactam ring of amp
Hygromycin B (HygB)
Blocks translocation from amino acyl site to peptidyl
site
Kanamycin (Kan)
Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by a phosphotransferase
Neomycin (Neo)
Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by a phosphotransferase
Streptomycin (Str)
Blocks protein initiation complex formation and
causes misreading during translation
Tetracycline (Tet)
Binds to 30S ribosomal subunit and inhibits protein
synthesis; tetr gene encodes a protein which prevents
transport of tet into the cell
Construction and screening of Library
• Why required?
• What is this library content?
• How to find the book from this library if it is
huge collection of books?
•Gene libraries
•cDNA libraries
•Library screening
Eukaryotic gene organization
enhancers
silencers
Genomic
library
construction
Partial Digestion
Screening a genomic
library using DNA
hybridization to a
(radio-)labeled DNA
probe
Note: a cDNA is commonly
(radio-)labeled and used as
a DNA probe to screen a
genomic library
Production of a (radio-)labeled DNA probe by the random primer
method [uses the Klenow fragment of DNA polymerase]
5’
3’
5’
3’
3’
5’
The first step in making a
cDNA library: Purification
of polyadenylated mRNA
using oligo(dT)-cellulose
Note: selection of the
proper source (organ,
tissue) of the RNA is
critical here!
Complementary DNA or
cDNA cloning:
cDNA library construction
Note: ds cDNAs are typically
placed in a cloning vector such
as bacteriophage lambda (l)
or a plasmid
Bacteriophage l
cloning system
Bacteriophage l cloning system
Cos sites
at the left
and right
ends
Cloning
site
Screening a cDNA
library using DNA
hybridization to a
(radio-)labeled
DNA probe
Screening a cDNA library with a labeled oligonucleotide probe
based on a known peptide sequence
Using polynucleotide kinase and
g-32P-labeled ATP to radiolabel oligonucleotide probes
Immunological screening of an
expression cDNA library with a
primary antibody and labeled
secondary antibody; note the
label is often an enzyme label
like alkaline phosphatase or
horseradish peroxidase, but it
can also be 125I
Animations for two related uses of
expression vectors
• Expression cloning of receptor proteins-see MCB Chapter 5
•
http://bcs.whfreeman.com/lodish7e/#800911__812046__
• Looking for protein-protein interactions with the yeast two
hybrid system-see MCB Chapter 7
•
http://bcs.whfreeman.com/lodish7e/#800911__812055__
Plus/min (+/-)
or differential
screening
A cosmid cloning system:
another possible cloning
vector which can be used
for genomic library but
not for cDNA libraries
In summary, you have seen:
•How to make and screen gene libraries
•How to make and screen cDNA libraries
•Several different cloning vectors including
plasmids, bacteriophage lambda (l), and
cosmids
How you will know the plasmid or DNA
you chosen the correct one?
• In case of DNA
• In case of RNA
• In case of Protein
Southern Blot Hybridization
Northern Blot Hybridization
•
RNA blots are called northern blots in recognition of the fact that the procedure is
analogous to the Southern blotting technique, but with RNA molecules being
separated and transferred to a membrane.
Analysis of RNAs by Reverse Transcriptase-PCT
(RT-PCR)
Analysis of Proteins by Western Blot
Techniques
END of First Part