Eukaryotic Gene Expression Flashcards
Transcription and Translation in Bacteria
- transcription and translation are coupled
- DNA is attached to the inner membrane so is accessible to everything in the cytoplasm
- transcription and translation can occur at the same time on the same molecule
Transcription and Translation in Eukaryotes
- transcription and translation take place in different locations
- transcription in the nucleus
- translation in the cytosol
- transcription and translation cant occur at the same time on the same molecule
- amplification can occur at both transcription and translation, one DNA sequence can be transcribed by many RNA polymerases and one mRNA can be translated by many ribosomes
Gene Expression Control Checkpoint
- Transcription Control
- Post Transcriptional Control
- Translational Control
- all 3 are used together to regulate gene expression
Gene Expression Control Checkpoint
Transcription Control
Positive: RNA polymerase recruited to specific gene to initiate mRNA synthesis
Negative: transcription of a specific gene is repressed
Gene Expression Control Checkpoint
Post-Translational Control
- processing of transcripts
- transport / sequestration of transcripts
- stability of transcripts (i.e. how long they exist in the cell)
Expression of an ‘Eclectic’ Eukaryotic Gene
- from 5’ to 3’ end, promoter, exons and introns, terminator
- gene is transcribed to form the primary transcript this removes the promoter and terminator and adds a poly-A tail
- primary transcript is spliced which removes introns
- this produces mature mRNA, 5’ utr, exons, 3’utr, poly-A tail
- translation removed 5’utr and 3’utr sequences and forms the precursor polypeptide
- processed to form the final polypeptide product
Eukaryotic Genes
- interrupted
- contains coding and non coding sequences
Promoter
-regulatory sequence ‘upstream’ of the coding sequence, made up of:
UAS - upstream activating sequence, binding site for transcription factor which is necessary to recruit RNA polymerase
PB - binding site for RNA polymerase
TS - transcription start site
Terminator
-regulatory sequence ‘downstream’ of the coding sequence signals that RNA polymerase should cease transcription and contains the poly-A signal required to modify eukaryotic mRNA
Exons
- sequences within a gene that are expressed in the mature mRNA
- typical exon sequence: GU—sequence—AG
Introns
- intervening non-coding sequences that interrupt the coding sequences of the DNA
- are transcribed in the primary transcript but are removed by splicing in the production of the mature mRNA
Alternative Splicing
-can generate multiple mRNAs (and therefore proteins) from the same DNA sequence
Splicing
removal of introns and putting together of exons
utr
- untranslated sequences present in the mature mRNA but not in the polypeptide
- can contain sequences that determine the efficiency of translation an stability of mRNA within the cell
5’ utr
-sequence at the 5’ end of the mRNA lies upstream of the translational start codon, untranslated when the mRNA encounters a ribosome
3’ utr
-sequence at the 3’ end of the mRNA follows the translation stop codon and is untranslated by ribosomes
Primary Transcript
-the first ‘RNA version’ of the gene that results from transcription, contains introns that are later removed by splicing
Enhancer
- a ‘volume control’ sequence
- a sequence found near a gene, causes greater quantities of surrounding genes to be transcribed
- thought to work by relaxing the chromatin around it
- not all genes are associated with an enhancer
- one enhancer may be associated with multiple genes
Cap
- a 7-methylated G residue that is added to the 5’ end of the transcript the ‘wrong way round’ (i.e. 5’ to 5’)
- gives structural rigidity to the 5’ end
- the 40S subunit of a eukaryotic ribosome binds to the cap to initiate translation
- other regulatory proteins can also bind to the cap
Poly-A Tail
- long stretch of A residues added to the 3’ end of the transcript by the enzyme poly-A polymerase
- contributes to stability after spliced RNA is transported to the cytosol
- -in the cytosol RNA is degraded either by 5’ to 3’ or 3’ to 5’ exonuclease activity
- -if it is 3’ to 5’ then the poly A signal has to be broken down before the protein sequence is damaged
- -length of poly-A signal is proportional to the stability of an RNA molecule in the cytosol
- can interact with cap via poly-A binding protein to facilitate recruitment of more ribosomes
What is gene expression a consequence of?
synthesis AND expression
Mendel and Molecular Biology
Description
- R gene (round) has been cloned and sequences
- it encodes a starch building enzyme
- when the enzyme is funtional in a cell (RR or Rr) starch grains fill the seed and when the seed dries it remains round
- when the enzyme is non-functional (rr), starch doesn’t fill the seed so as it dries it becomes wrinkled
- the phenotype is only visible when the seed dries out
Mendel and Molecular Biology
When is the R gene expressed?
- when transcription begins?
- when mRNA is present in the cell?
- when the protein is present in the cell?
- when the enzyme is active in the cell?
- when the phenotype is observable
Transcriptional Control
Positive - how RNA polymerase is recruited to specific genes to initiate mRNA synthesis
Negative - how transcription of a specific gene may be repressed
Promoter and Gene Expression
-the promoter is a key sequence in a gene for the regulation of expression
Surrogate Genetics
Why?
-as we can’t do the same experiments in vivo with eukaryotes as we do with bacteria which can quickly produce massively large numbers of offspring
Surrogate Genetics
Steps
1) clone gene
2) mutate in vitro either randomly or directedly OR sub clone fragments into ‘probe’ vectors
3) reintroduce into a test-bed host
4) measure gene exxpresssion
Promoter-Reporter Fusion
Principle
- clone the promoter region of your gene of interest and insert immediately adjacent to a ‘reporter’ gene
- the reporter gene has a product which is easily assayed
- introduce the promoter and reporter gene into a suitable host
- measure accumulation of the reporter gene product
Examples of Reporter Genes
LacZ - beta-galactosidase
GUS - beta-glucuronidase
LUC - luciferase
GFP - green fluorescent protein
Lac Z and GUS
- both from E.coli
- release galactosidases and glucuronides from colourless substrates resulting in the formation of an insoluble indigo dye
- easy to see expression
- GUS is the most popular reporter gene for plant gene analysis as there is no homologous plant gene
North American Firefly
- cells in fireflies emit light as a sexual attractant
- this emission of light occurs as a result of the action of the enzyme luciferase on the substrate luciferin in the presence of ATP
- the gene encoding luciferase can be expressed as a transgene
- cells in which the gene is expressed can be visualised in vitro by providing them with luciferin
GFP
- green fluorescent protein
- comes from the jellyfish Aequoria Victoria
- a 238 amino acid protein that glows green when illuminated with blue or long wavelength ultraviolet light
- non-toxic and can be visualised in whole organisms or at the sub-cellular level
- there are many spectral variants of the jellyfish protein (that glow different colours) allowing multicolour imaging of tagged proteins in vivo
Promoter-Reporter Fusion
Stages
- isolate DNA from target cell
- digest DNA with different restriction enzymes
- find a combination of restriction enzymes that will digest the promoter out of the DNA
- ligate the promoter into a cloning site upstream of the reporter gene
- this promoter-reporter fusion has to be expressed in a cell similar to the organism that it came from with a compatible expression system
- introduce into suitable host/host cells by transformation
- assay for the reporter gene product
- important sequences in the promoter can be identified by deletion analysis (in vivo) and by DNA footprinting (in vitro)
Deletion Analysis - Heat Shock Example
- can identify regulatory elements in vivo, e.g. a heat shock promoter
- heat shock genes are only expressed above a certain temperature
- the promoter is 400bp long, the first 100, 250, and 325 base pairs were removed from the 5’ end of the promoter
- the promoter was inserted upstream of a LacZ gene
- at 25C there is no expression of LacZ
- at 40C there is 100% activity with the 400bp promoter
- there is the same activity for the 300bp promoter at 40C
- with the 150bp promoter there is 85% activity at 40C
- so there must be some level of gene expression regulation in the -150 to -300 region but not the heat inducibility
- with the 75bp promoter there is only 15% activity at 40C
- the control must be cis acting - in the dame piece of sequence as the gene
End Labelling of DNA
- incubate double stranded DNA with alkaline phosphate
- this replaced the 5’ phosphates with a 5’ -OH
- mix with the enzyme, T4 polynucleotide kinase, this enzyme takes the terminal phosphate from an ATP molecule and puts it on a polynucleotide
- this can only work if there isn’t a phosphate already at the end of the molecule
DNA Footprinting - A Nuclease Protection Assay
1) prepare end labelled DNA fragment
2) extract nuclear proteins
3) mix (1) and (2) together
4) partially digest with DNaseIby keeping the concentration of DNaseI very low
5) DNase 1 is a nuclease that cleaves DNA to its component nucleotides
6) if mixed only with the DNA at low concentration it would produced fragments of all possible sizes
7) in the nuclear proteins mix there are transcription factors, they will bind to the DNA at their complimentary sequence
8) when the mixture of DNA and proteins is digested with a low concentration of DNaseI, the regions of DNA covered by the transcription factors are protected
9) when you run the two samples on the agarose gel, there will be a gap on the gel corresponding to the sequence covered by the transcription factor
10) from here it is easy to identify the protected sequence
Summary of the Identification of the Regulatory Sequences in Eukaryotic Genes
-together in vivo promoter analysis and in vitro protein binding expreiments allow us to identify the regulatory sequences in eukaryotic genes
