Topic 3 - Structure of genes, their regulation & expression Flashcards
The major control point in prokaryotes and eukaryotes is…?
transcription initiation (turning genes on & off)
Control of transcription initiation is mediated by…?
promoters
Proteins that bind to promoters & stimulate/repress transcription are called…?
transcription factors
RNA polymerase is made up of how many subunits?
4 (2 alpha & 2 beta)
A transcription factor in prokaryotes?
sigma subunit
Sigma subunit leaves promoter during which phase?
elongation
Termination of transcription (prokaryotes) can occur in which 2 ways?
Rho-dependent - Rho factor pulls mRNA away from RNA polymerase
Rho-independent - stem-loop structure signals release of mRNA
What are inducers? In which organisms are they found?
factors that turn genes on/off in response to environmental cues in PROKARYOTES
What are some environmental cues that may affect prokaryotes…?
sugars nitrogen heat salt light intensity
T or F - operons are found in all organisms
false - not present in eukaryotes
What are operons? What structures do they contain?
2 or more protein coding genes transcribed into a single polycistronic mRNA -> translated into several different proteins at ribosomes
Operons contain: promoter (DNA), repressor protein, operator (DNA) & genes (DNA)
What are cistrons?
the several structural genes next to the operator
What are the cistrons in lac operon? What do they encode? What are their functions?
lacZ - beta-galactosidase (converts lactose -> glucose & galactose)
lacY - permease (transports lactose into cell)
lacA - acetylase (acetylates lactose)
Why is the operon mRNA said to be ‘polycistronic’?
Because it is translated into several proteins
What are the 3 different operons…? How many cistrons in each?
Lactose utilisation operon (lac operon) 3 cistrons Histidine biosynthesis (his operon) 11 cistrons Tryptophan biosynthesis (trp operon) 5 cistrons
Who discovered the lac operon?
Jacob & Monod
What does the lac operon do?
breaks down lactose (milk) eg. rumen bacteria of calves
Lac operon is said to be in ‘off’ position when lactose is …?
absent - lac genes can not be expressed
What is the repressor gene? What does it do? When is it present?
lacI -> mRNA -> repressor protein
expressed all the time
In the lac repressor, what are the 2 domains?
DNA-binding domain (near N terminus)
Inducer-binding domain (near C terminus)
Where does the lactose sugar interact on the lac repressor?
Inducer-binding domain (near C terminus)
When lactose enters cell & binds to repressor, is this +ve or -ve control?
-ve control
What is +ve control in lac operon?
glucose absent -> ++cAMP -> cAMP binds to CAMP receptor protein (CRP or CAP) -> cAMP-CRP complex -> lac promoter -> transcription
End product of lactose metabolism?
glucose (-ve feedback inhibition) as ++glucose -> decreased cAMP
How does the trp operon function…?
When tryptophan is present -> trp binds repressor (repressor active) -> binds operator -> transcription blocked
Opposite to lac operon function
What is araC?
positive regulator for all arabinose genes -> break down sugar arabinose
When arabinose is not present…?
AraC dimer binding at araO & araI sites -> no araBAD genes transcribed
What does RNA polymerase I transcribe?
rRNA genes
What does RNA polymerase II transcribe?
protein coding & micro RNA genes
What does RNA polymerase III transcribe?
tRNA & small RNA genes 5S
Where does rRNA processing occur?
nucleolus
Which rRNA units make up ribosomes?
28S rRNA & 5.8S rRNA - large ribosomal subunit
18S rRNA - small ribosomal subunit
Eukaryotic gene expression is controlled at 8 stages. What are they?
chromatin remodelling transcription initiation mRNA (transcript) processing transcript export to cytoplasm transcript stability translation protein processing, folding & transport protein stability, degradation & modification
Draw diagram of control points
slide 26
Reasons for extensive control in eukaryotes…?
Large genomes (extensive non-coding regions)
compartmentalisation
transcript processing (intron/exon structure)
no operons
proteins can be products of several genes (multi-subunit eg. insulin, haemoglobin)
enhancers & silencers
cell type specific expression necessary
Re. transcriptional control of pol II genes via basal factors - what are they?
TATA box binding protein (TBP)
TBP associated factors (TAF)
How do basal factors control pol II genes?
basal factors bind to promoter, then RNA pol II binds to basal factors
What are cis-acting sequences? Where are they found? An e.g’s?
regions of DNA sequences that bind proteins which interact with RNA polymerase -> regulate transcription
Found in region of promoter or far away, but on SAME CHROMOSOME
eg. enhancers, lac operon
What are trans-acting factors? e.g?
proteins that bind to cis-acting sequences distant from gene from which they were transcribed
transcription factors
Basal factors + pol II bound to DNA = ?
low-level transcription
Basal factors + pol II + activator protein bound to DNA = ?
high-rate of transcription
2 common motifs…?
helix-loop-helix
zinc finger-leucine zipper
Which part of the leucine zipper is the DNA-binding domain?
the zinc finger part
How do steroid hormones (SH) control transcription?
SH binds to steroid hormone receptor -> allosteric change in receptor -> SH receptor & SH -> bind enhancer -> + transcription
Transcription factors have a … structure?
modular structure - ie. have DNA binding domain separate from the activator domain
DNA is kept within the cell via …?
chromatin ‘beads on a string’
Structure(s) of chromatin…? This complex is called …?
2 helix loops wound around 4 histone proteins -> nucleosome
Why is chromatin the cells normal DNA storage form?
Because it reduces binding to basal factors & RNA pol II to very low levels
What do chromatin remodelling proteins do?
unwind DNA helix loops from histone to expose promoter so that basal factors can bind -> transcription
What are ‘silenced’ genes/heterochromatin?
when DNA methylation in a region causes hypercondensation in nucleosomes -> completely inactive genes
What is alternative splicing?
Different cell types process same pre-mRNA differently -> 2 different proteins from same gene
Example of alternative splicing…?
- Calcitonin mRNA in thyroid 32aa for calcium balance
- Calcitonin gene related peptide (CGRP) mRNA in hypothalamus 37aa vasodilator
What is translation controlled by?
protein synthesis initiation factors & inhibitors
Re. protein mRNA stability & half lives. Tubulin, insulin receptor, pyruvate kinase (hrs)?
tubulin (4-12hr) insulin receptor (9hr) pyruvate kinase (30hr)
How many micro-RNAs identified & characterised?
2000-2005
- 1/4 from introns protein coding transcripts
- 3/4 from products of primary transcripts devoid of ORFs
- may exceed number of protein coding genes (>30000)
How do micro-RNAs work?
trans-acting single stranded micro-RNAs regulate eukaryotic gene expression
Structure of miRNA’s…?
60-120 ribonucleotides long segments of complimentary reverse sequence- hairpin stem-loop structures
Applications of miRNA
method for developing powerful new RNA-based therapies for treatment of disease
miRNA are processed by which 3 complexes? Where are they found?
- Drosha - cropping (nucleus)
- Dicer - dicing (cytoplasm)
- RISC - miRNA degradation (cytoplasm) => miRNA
How do miRNAs interfere with transcript stability or translation…?
- mRNA cleavage (miRISC binds to mRNA in perfect complementarity) => mRNA breaks down
- Translational repression (2 miRISC binds to mRNA for incomplete complementarity)
What is post-translational control?
modifications that occur to proteins (after translation) that give them functionality
What are some types of post-translational control?
Modification
- glycosylation (addition of sugar)
- phosphorylation (addition of phosphate groups)
Removal of AAs
Assembly of proteins
Import into organelles (eg. nucleus, mitochondria)
Protein degradation (ubiquitination) eg. dissolving glue protein during anaphase so sister chromatids can seperate
Ubiquitination example in more detail…?
At metaphase - sister chromatids bound together by glue proteins
At anaphase - expression of anaphase promoting complex (APC) -> attracts ubiquitinating enzyme -> adds ubiquitin to glue protein -> recognised by proteosome which dissolves glue protein -> sister chromatids can separate and continue through anaphase
Phosphorylation example…?
adrenaline binds to beta-adrenergic receptor -> allosteric change -> ++HR etc. -> kinase transfers phosphate -> receptor -> Adr is removed -> phosphatase removes phosphate
Re-draw control points for eukaryotic gene expression summary diagram
slide 51
