Lecture 18 - Protein expression Flashcards
DNA stores information (genes), which is expressed as
RNA and proteins
The information (genes) is expressed in 2 steps
- A copy of RNA is made in a process called transcription
- The copied information in the form of mRNA is translated to proteins in a process called protein synthesis or translation
Transcription begins near
promoter sites
Promoter sites are
docking sites of RNA polymerase to initiate transcription
Transcription ends near
termination sites
RNA polymerase synthesizes a
terminator sequence
mRNA in eukaryotes is modified after
transcription
In bacteria, polypeptide chains are encoded by a
continuous DNA sequence
Introns
intervening base sequences that do not appear in the mRNA of that gene product
Exons
DNA sequences that are expressed
There is a consensus sequence in higher eukaryotes that signal
splicing
Splicing only occurs in the
nucleus and only in eukaryotes
The consensus sequence in higher eukaryotes is a _____ at the 5’ end and a _____ at the 3’ end
GU; AG
All introns have a ___________ 18 to 40 nucleotides upstream from 3’-splice site (pyrimidine tract)
branch site
How does the sequence of mRNA translate into the sequence of a protein?
The ribosomes can “read” the genetic code embedded in the mRNA
What is the genetic code?
The relation between the sequence of bases in DNA (or its RNA transcripts) and the sequence of amino acids in proteins
Features of the genetic code (4)
- Three nucleotides (a codon) encode one amino acid
- The code is non-overlapping
- The code has no punctuation
- The code is degenerate
What is the Shine-Dalgarno sequence
A ribosomal binding site in bacterial and archaeal messenger RNA, generally located around 8 bases upstream of the start codon AUG
Techniques used to control protein expression (3)
- Can be tagged in vivo with fluorescent proteins to study localization
- Can be purified to study their structure, interactions, and functions
- Can be used as medicine
General strategy of protein expression consists of
- Tranfecting/transforming/transducing cells with DNA of choice
- Transcription, translation (and modification) of protein of interest
- Purificatino and/or studying protein of interest
Mammalian systems can express proteins both
transiently and through stable cell lines
Insect cells can be used to produce ____________________ with the correct post-tranlational modifications
complex eukaryotic proteins
Two types of insect expression systems:
- Baculovirus infected
- Non-lytic insect cells
Bacterial cells can produce vast quantities of
proteins rapidly and cheaply
Plant expression systems provide a cheap and low-tech means of
mass expression of recombinant proteins
Yeast expression systems can generate
large quantities of recombinant eukaryotic proteins
Proteins are assembled in vitro using
purified components of the transcription and translation machinery
Expression vectors are optimized for the production of
large amounts of proteins
The gene of interest in the expression vector often contains fusion tags to
easily purify the expressed protein
Promoter sequences are designed to
drive the transcription of large amounts of a protein-coding gene
Expression vectors often contain a polylinker region that includes
several restriction sites
Expression vectors contain (6)
- Promoter
- Fusion tag
- Polylinker
- Ribosomal binding site
- Termination sequence
- Start and stop codons
pET vectors are commonly used to
express proteins using the T7Lac system
In E. coli, metabolism of lactose is regulated by the
lac operon
In the lac operon, the presence of lactose starts
expression of the β-galactosidase gene
An operon is a
functioning unit of DNA, which contains a cluster of genes under the control of a single control site
The lac operon encodes genes that are required for
the transport and metabolism of lactose
The regulator gene (lacl) encodes
a repressor protein
The promoter sites (p) directs the
RNA polymerase to the correct transcription initiation site
In the absence of lactose, the lac operon is
repressed
Allolactose relieves the
repression of the operator
The inducer (allolactose) triggers gene expression by
preventing the lac repressor from binding the operator
Allolactose binds to the
lac repressor
