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Lecture 8
A toolbox for mechanistic biologists
(continued)
Basics molecular genetics
Mass spectrometry
measures
m/z
(mass/charge)
Complete sequencing of a protein can be accomplished using MS/MS in
conjunction with genomic information
What is an ideal situation for mechanistic studies of a protein of interest?
(Chapters 3 and 5)
Prokaryotic expression system
Eukaryotic expression system
Isolate that protein, generate antibodies, localize it in the
cell, find counterparts, study it in vitro!
Two different types of
zonal centrifugation:
1. Velocity sedimentation
2. Equilibrium
centrifugation in gradients
Density!
Myc- or 6His-tag affinity chromatography
6His-tagged proteins
can be eluted with
imidazole (His analog)
from a Ni-NTA column
Identify the main product on a western blot and other
components (subunits) that can be co-purified
Restriction enzymes cleave specific DNA sequences,
many of them produce ‘sticky ends”
If the gene is known (present in the database), then it can be amplified
and cloned by PCR (Fig. 5-23)
The fragment can be amplified with primers that contain restriction
enzyme cleavage sites, then ‘trimmed’ and inserted into a plasmid
(expression vector) Fig. 5-24
CLONING IN BACTERIA
Fig. 5-13
Blue – exons (coding regions)
Green – introns (non-coding regions, which will be spliced out of the mRNA)
Unlike in yeasts, in higher eukaryotes genomic DNA does not represent
continuous coding regions, thus spliced mRNA is usually taken as a starting
material
Cloning from higher eukaryotes uses mRNA and involves a reverse
transcription step (RT-PCR)
RNA → DNA transcription
If RT-PCR was performed with sequence-specific primers, then the trimmed
fragment of interest can be inserted directly into an expression vector.
If RT-PCR was done with generic (non-specific) primers, then a cDNA library
can be created and probed against known or predicted sequences.
Cloning into a plasmid vector
Cloning into lambda phage
Libraries can be
screened by
hybridization to a known
oligonucleotide probe
(see Fig. 5-16)
Basic Molecular Genetic Mechanisms
Bottlebrush pattern of
nascent ribonucleoprotein
complexes transcribed from
DNA by RNA polymerase I
Fig. 4-1
The structure of DNA
(4-2, 4-3)
B DNA
Double stranded DNA can exist in two other forms: A and Z DNA (Fig 4-4)
Single- and Double-stranded DNA can be distinguished by OD260
The melting (thermal strand separation) temperature depends on
the GC content
DNA is bendable
DNA has regulatory regions (promoters, enhancers)
and coding regions (open reading frames)
Binding to the promoter is assisted by transcription factors
The rate of transcription at
37oC is ~ 17-20 nucleotides/s
(1000 per min)
RNA polymerase is extremely
processive, i.e. it remains
stably associated with the
DNA template all the way to
the stop site.
Intron-Exon structure of the Fibronectin gene and alternative splicing
Types of RNA are involved in protein synthesis:
1. Messenger (mRNA)
2. Transfer RNA (tRNA)
3. Ribosomal RNA (rRNA)
In Eukaryotes:
RNA polymerase I - transcribes
precursors 28S, 5.8S and 18S
rRNAs
RNA polymerase II - transcribes
all protein-coding genes
RNA polymerase III - transcribes
tRNAs, 5S rRNA and small RNAs