WHOLE MODULE Flashcards
What is meant by the term isoform
An isoform is a protein variant that differs based on posttranscriptional modifications
What process is used to create different protein isoforms
Alternative splicing
What regions of the immature mRNA transcript are removed during splicing
Introns
Splicing means that different proteins can be created from the same gene, T or F
T
All eukaryotic genes contain introns and exons, T or F
F – yeast do not contain introns
What components of genes allow for alternative splicing to occur
Optional introns and exons, mutually exclusive exons and internal splice sites
40% of the Drosophila genome is alternatively spliced. What percentage of human genes are also spliced
0.75
Other than splice donor and acceptor sites within gene transcripts, what other features of the mRNA allows for alternative splicing
Other sequences contained within the mRNA and the secondary structure also affects the choice of splice sites
How is splicing regulated
By RNA binding proteins
Give an example of a gene that undergoes alternative splicing
Dscam in Drosophila is a very large gene that produces a massive mRNA transcript containing 100 exons. The final mature mRNA will contain one exon from 12 A exons, 48 B exons, 33 C exons and 2 D exons, creating 38,000 splice variants of the dscam gene product. This is indicative of its role in the Drosophila nervous system
What are the three key genes in determining sex in Drosophila and what are their roles
Sex lethal (sxl), a RNA binding protein and splicing repressor, transformer (tra), a RNA binding protein that acts as a splicing activator and, doublesex (dsx) a transcription factor.
How are male Drosophila determined using alternative splicing and the interactions between the three sex determining genes
Male Drosophila have one X chromosome and this acts as the default pathway for sex determination in fruit flies. The transcripts for sxl and tra are spliced to give rise to inactive proteins. The dsx transcript is also spliced but this gives rise to a male specific transcription factor that acts as a transcriptional repressor of female-specific genes.
How are female Drosophila determined using alternative splicing and the interactions between the three sex determining genes
Female Drosophila have two X chromosomes and a sex chromosome to autosome ratio of 1. The presence of two X chromosomes results in the transient activation of an alternative sxl promoter sequence which leads to the production of the sxl transcript which is then spliced and translated to form a splicing repressor. The sxl protein produced binds to other sxl transcripts and represses splicing by blocking binding of U2AF. This feeds back to result in more production of functional sxl transcripts. The sxl protein also binds to the tra transcripts causing an alternative splice that produces a functional tra protein after translation. The functional tra protein is a splicing activator and causes splicing of the dsx transcript. Splicing of the dsx transcript produces the female dsx transcript which is translated to the female dsx isoform. The female dsx protein is a transcriptional repressor of male specific genes.
Give an example of how polyadenylation can act as a regulation of gene expression
The site of polyadenylation within the mRNA can be regulated. B lymphocytes for example can produce two different isoforms of an antibody. The antibody gene for a specific antigen has two possible positions for cleavage and polyadenylation. This determines whether the antibody is to be secreted or to remain membrane-bound. To produce the membrane-bound antibody, the cell produces the long transcript of the antibody. In this case the first stop codon within the antibody mRNA transcript is spliced out. This results in the translation of the transmembrane domain. Once the protein is secreted it remains tethered to the membrane via the transmembrane domain. For an antibody to be secreted the short transcript is produced which results in the loss of a splice acceptor site. Thus, no splicing of the transcript occurs and the first stop codon isn’t lost. This results in a termination of translation at the first stop codon, prior to the transmembrane domain region. This means that when the antibody is secreted it isn’t tethered to the membrane by a transmembrane domain.
What is meant by the term leaky scanning
Sometimes the first AUG codon can be missed by the ribosome
Sequences around the start codon help to initiate translation, T or F
T
What is meant by the Kozak sequence
The Kozak sequence is the optimal translation initiation sequence that contains the start codon and ideal bases adjacent.
Recall the Kozak sequence
ACCAUGG
How can leaky scanning lead to the production of different protein products
If the sequence is less than optimal the ribosome can miss the first start codon and begin at the second or third AUG. These proteins will all be produced in the same reading frame, differing only by the sequence in the N-terminus
High levels of what translation cofactor increase the probability that the first start codon will be recognised
eIF-4F
How does the HIV virus make use of regulated nuclear transport to influence gene expression
After integration of the HIV genome into the host cell, the whole genome is transcribed as one piece of mRNA. Alternative splicing allows for the many different protein products to be made. Although the full-length mRNA is needed to make new virions the unspliced mRNA containing the entire virus genome cannot leave the nucleus. One of the proteins encoded by the virus genome is the rev protein. During the early stages of infection when only the alternatively spliced viral mRNA can leave the nucleus, the rev transcript moves through the nuclear pore and is translated. The rev protein then interacts with the nuclear pore in late stage infection to allow the exit from the nucleus of the unspliced mRNA.
Signals within which regions of mRNA transcripts target them to particular parts of the cell
3’ and 5’ untranslated regions
How are 3’UTRs recognised by cellular proteins which lead to the sequestration of RNA in one part of the cell
Intermolecular base pairing within the 3’UTRs form stem loops which are recognised by cellular proteins
What is the role of ferritin in the regulation iron availability
Ferritin is a protein that stores iron inside the cell leading to a decrease in Fe availability
What is the role of transferrin in the regulation iron availability
Transferrin is a receptor that imports iron into the cell leading to an increase in available Fe
How does aconitase interact with ferritin and transferring mRNA
Aconitase binds to stem loops in the 5’UTR of ferritin mRNA and in the 3’UTR of transferrin mRNA
Aconitase can also bind to iron itself, T or F
T
Explain the role of aconitase in increasing Fe availability when iron levels are low
Aconitase binds to stem loops in the 5’UTR of the ferritin mRNA and blocks translation by physically blocking the ribosome from moving along the transcript. Aconitase also binds to stem loops in the 3’ UTR of transferrin mRNA and blocks its degradation. Binding of aconitase stabilises the transferrin mRNA thus increasing transferrin synthesis. Decrease ferritin translation and increase transferrin stability and synthesis results in an increase in intracellular [Fe]
Explain the role of aconitase in decreasing Fe availability when iron levels are high
Aconitase binds to iron itself in the cytoplasm which causes a change in its conformation. A change in aconistase comformation causes it to dissociate from the stem loops in the 5’UTR of the ferritin mRNA and the 3’UTR of the transferrin mRNA. The now unstable transferrin mRNA transcript is then degraded quickly and the ribosome is now free to move along and translate the ferritin mRNA. This leads to a decrease in Fe availability
What co-factor of the translation machinery is required for all mRNA translation
eIF-2
When a cell is in the G0/quiescent phase of the cell cycle, globally, translation is turned down. How is this achieved
Phosphorylation of eIF-2 causes its tight binding to eIF-2B. eIF-2B is usually required as a guanine nucleotide exchange factor but tight binding of eIF-2B to the phosphorylated eIF-2 prevents it’s recycling by GTP displacing the bound GDP. By preventing guanine nucleotide exchange, eIF-2 is prevented from initiating translation.
eIF-2 with its bound GTP binds to Met-tRNA to start ribosome scanning, T or F
T
What are IRES sequences and how can they influence gene expression
Internal ribosome entry sequences are stem loops contained within RNA that can initiation formation of the ribosome independent of the Cap/PolyA initiation complex by mimicking it.
IRES translation initiation is an effective way of encoding a second reading frame within RNA, T or F
F – this is not a very effective method with less than 10% of second ORF translation
Which translation initiation cofactor is required to bind to the IRES stem loop and initiate translation independent of the 5’-Cap and PolyA tail
eIF-4G
Where are IRES sequences commonly found
IRES sequences are often found in viral transcripts
How can viruses use IRES sequences to promote translation of their transcripts
Viruses favour translation of their transcripts by cleaving eIF-4G in a way that prevents it from binding to eIF-4E but can still bind to IRES stem loops
What is the significance of eIF-4G cleavage in apoptosis
eIF-4G is cleaved during apoptosis to prevent its binding to eIF-4E and hence prevent any more translation
RNA stability is defined by the half-life of the different mRNAs and varies greatly, T or F
T
How does the polyA tail change over time and act as a clock to determine mRNA age
The polyA tail usually starts at around 200 residues in length but as time goes by this is gradually degraded by exonucleases. Once it reaches 30 nucleotides in length it is de-capped and degraded. Hence the number of adenines in the PolyA tail can determine the age of the mRNA transcript
How can mRNA half-lives be extended
Re-adenylation of the polyA tail
Often factors that promote translation also promote mRNA degradation to prevent overexpression, T or F
F – de-adenylating nuclease (DAN) is responsible for de-adenylation of polyA tails and competes with eIF-4E binding to the mRNA cap.
What technique is used to visualise protein localisation within a cell or tissue
Antibody staining
What technique is used to visualise gene transcription inside cells and tissues and is quick and inexpensive
In situ hybridisation
GFP and transgenics are techniques used to visual gene expression and protein localisation, T or F
T
Which method is often the first port of call to visualise RNA expression due to its cheap costs and short time
In situ hybridisation
Explain how an expression plasmid is made in the process of making an antibody
The mRNA for the protein which you want to target an antibody for is extracted from a cell and converted to cDNA. This cDNA sequence is inserted into a vector containing a bacteriophage promoter. The cDNA is incorporated into the expression plasmid next to the bacteriophage promoter which is included as these promoters drive high levels of RNA synthesis and hence will produce large amounts of the protein. The promoters are also modified so that they are inducible either by chemical exposure or temperature changes to induce gene expression. These expression plasmids also contain an epitope tagging system to allow for rapid and efficient purification of the protein. These tag coding sequences are inserted in-frame and upstream of the protein of interest cDNA and are sequences to which antibodies are readily available for. These expression plasmids are then injected into bacteria which can then make the protein.
Why are expression plasmid promoter regions made to be inducible
The problem with incorporating bacteriophage promoters is, due to their high levels of expression, the bacteria in which these vectors are introduced tend to die quickly due to exhaustion. By making the promoters inducible you can minimise the time spent synthesising protein to allow the cells to survive
Explain the purpose of an epitope tagging system when creating expression plasmids in antibody synthesis
Epitope tags are short coding sequences integrated upstream of the cDNA in frame. These will be transcribed and translated with the desired protein and allow for the rapid and efficient purification of the protein. They code for peptides to which antibodies are readily available and already manufactured for.
Explain the process of antibody-affinity purification
The crude extract is poured onto a column containing beads with antibodies for the epitope tag bound to them. A pH 7 buffer is then also loaded into the column. The crude extract then runs through the column and the protein of interest is retained by binding of the antibody covered beads to the epitope tag. The rest of the crude extract travels through the column and is removed. The column then undergoes a series of subsequent washes with the pH 7 buffer until no more protein comes out of the column. The pH is then reduced to pH 3 with another buffer which breaks the interaction between the antibody and the epitope tag and protein of interest. This results in elution of the pure protein from the column.
Once the target protein, for which you want to create an antibody for, is isolated from the crude extract, how is this then used to make the specific antibodies
The purified protein is injected into an animal (i.e. rabbit, mouse, donkey) several times, typically once a month for a three-month period. After 3 months, the blood is taken from the animal and the antibodies are then purified.
What is the name of the region of a protein to which an antibody binds to
Epitope
How are antibodies visualised once they are bound to a target protein
Tagging antibodies with dyes or enzymes to allow determination of where proteins are localised
What is meant by antibody sandwiches and why are they used to visualise protein localisation
Antibody sandwiches are produced by raising a secondary antibody that will bind to the first antibody. This produces an antibody sandwich of the primary antibody bound to the target epitope and then a secondary antibody bound to the first one. The secondary antibodies are usually tagged and this allows amplification of the signal. Because more than one tagged secondary antibody will bind to the primary antibody, a greater signal is produced
How are tagged secondary antibodies produced
Secondary antibodies are raised against general antibodies from the original animal species in which the primary antibodies were raised. These secondary antibodies produced in a different species will bind to any antibodies from the other species. These are then conjugated with dyes that are fluorescent allowing for the detection of protein location using specific wavelengths
Give two examples of commonly used enzyme conjugates in antibody detection
Alkaline phosphatase – turns the substrate blue. Horseradish peroxidase – turns the substrate blue
To stain a cell/tissue with a tagged antibody, the animal/cells must be chemically fixed, how is this achieved
A fixative, usually formaldehyde is introduced to cross-link proteins together
What are the two types of antibody staining and what are they used for visualising
Whole mount staining is used to visualise whole structures and tissues whereas staining on section involves antibody staining of slices creating cross section, this is often used in human samples.
Explain the process of in situ hybridisation
Start with a purified vector containing the cDNA of interest known as the template. This cDNA is incubated with RNA polymerase to make an antisense RNA probe. The antisense RNA probe will have incorporated epitope tagged nucleotides, special nucleotides with epitope tags conjugated to them. The cells are then incubated with antisense probe which will hybridise with the endogenous mRNA. Excess probe is washed off. The epitope tags often involve alkaline phosphatase which allows the mRNA to be visualised
Explain the differences seen in bicoid mRNA and protein localisation seen by using antibodies and in situ hybridisation
In situ hybridisation of bicoid mRNA will reveal its localisation in cells at the anterior region of the Drosophila embryo with defined borders. However the antibody staining for the bicoid transcription factor protein will show a different pattern. It would show a decreasing gradient of the bicoid protein, indicative of a morphogen
How is bicoid mRNA localised anteriorly in the Drosophila embryo
Contains within its 5’UTR a region that localises it at the anterior region of the cells
Explain how GFP works to provide fluorescence
GFP is excited by blue light with a wavelength of 475nm. Excitation of the GFP results in electrons within the protein increasing their energy level. The transition of these electrons back down to low energy states gives off energy in the form of light. For GFP, this energy emits is given off as green light with a wavelength of 510nm
Fluorescence always gives off an emission wavelength that is greater than the excitation wavelength and a lower energy, T or F
T
Explain how GFP is used in gene fusion to create transgenic lines
Firstly, you genetically engineer GFP onto the end of the last exon of the target protein by adding a GFP encoding sequence to the end of the last exon. This will be translated to a fusion protein contiaining the target protein and a fused GFP protein.
What does GFP gene fusion specifically allow that reporter constructs done
Because a functional protein of interest is produced the subcellular localization of the protein once can be studied
How does the incorporation of GFP still lead to the production of a functional protein
GFP doesn’t interact with the other protein and doesn’t impact its folding. Hence a functional protein is produced which allows for the study of where it is localised. This method only tells you were and when gene is expressed because no functional protein is produced other than GFP
Once GFP constructs have been created how are these then used to visualise protein expression
Microinjecting a solution of the DNA into the one-cell zygote is followed by incorporation of the construct into the host genome. The DNA randomly integrates into the genome by the DNA repair machinery and leads to the creation of a transgene
What is the name given to the representation of the differences between species and their evolution from common ancestors
Phylogenetic tree
It has been shown that most of the genes across kingdom Animalia have been relatively conserved. How has this been achieved
BLAST analysis of protein structures to determine regions of significant homology
It was determined that what makes members of the animal phyla different is not differences in the genetic sequence as such but what difference was seen
Changes in expression of a common set of genes
For great apes and man the change in the nucleotide sequence is about 1% every 10million years, T or F
T
If the human and great ape nucleotide sequence changes by roughly 1% every 10 million years and the common ancestor of humans and chimpanzees evolved 5 million years ago, how different are their genomes. Represent this as a fraction and as a number of nucleotides
0.5% difference – 1 in 200 nucleotides
Firstly, molecular data is used to distinguish specices which is then backed up by morphological data, T or F
F – vice versa
When assembling the phylogenetic tree a specific gene is used to determine relation, this gene is FOXP2. Why is FOXP2 used as a way of differentiating between species
FOXP2 is a highly conserved protein which only shows differences at a few positions in the amino acid sequence
Which positions in the amino acid sequence does the FOXP2 vary
80, 303 and 325
Mice and chimps both have a threonine residue at position 303, what therefore can be inferred about the common ancestor of these species
Their common ancestor must have also had a 303T
Humans and chimps both have 80D in the FOXP2 gene meaning that the common ancestor between these species also possess an 80D. Which amino acid is denoted by D
Aspartate
When sorting animals into phyla, programmes similar to BLAST consider all possible relations between animals. How are they then sorted
The tree is assembled based on the simplest model with the fewest changes
What is meant by the term parsimony
The idea that you always assume the simplest model
Convergent evolution goes against the parsimony model. How does convergent evolution account for differences in the amino acid sequence
Changes in the amino acid sequence of two animals occur independently of each other
When assembling the phylogenetic tree, molecular phylogeny is compared with morphological phylogeny and fossil records to give us a deeper understanding of evolution, T or F
T
Although there are only 4 families of vertebrate FGF receptors, how many distinct receptors are there
22
The Ciona or sea squirt is a distant vertebrate ancestor that only contains 4 FGF receptors. What does this tell us about the common ancestor of Ciona and modern vertebrates
The common ancestor also had only 4 FGF receptors
What can explain the other 16 FGF receptors present in vertebrates that aren’t in sea squirts
These will likely have arisen due to genome duplication both locally and by ploidy events
What is the name given to a duplicated gene present in the genome
Paralogue
Chromosome duplications or duplication mutations are seen frequently when comparing genomes, T or F
T
When a gene is first duplicated, what is its role
It is redundant
Over time duplicated genes evolve allowing a refinement of function or for a new function to development, T or F
T
What ways can the extra copy of a gene change
Can change the pattern of gene expression or the structure/function of the protein
Big changes to protein structure are caused by what type of mutations
Domain swapping
Smaller changes in the protein structure of a duplicated gene are caused by nonsense mutations, T or F
F – missense mutations
What is considered to be the common driving force for morphological evolution of animals
Changes in expression of genes
What changes in the transcription machinery present within the DNA explain why expression pattern changes have a major role in morphological evolution
Enhancers can change easily
What process can account for bringing a new enhancer regions close to the coding sequence of a gene
Non-homologous recombination
Enhancers must occur upstream of a target gene, T or F
F – enhancers can occur anywhere in the gene sequence and their exact position is usually unimportant
Changes in protein structure have to be precise so as not to introduce a variety of negative effects. Give some examples of these effects
Introduction of a stop codon, change in the reading frame, interference with protein folding or a disruption in RNA splicing
Describe the differences seen in the development of appendages in the fruit fly and Crustacea
Crustaceans development limbs throughout their abdominal and thoracic segments whereas in Drosophila, no legs develop in the abdominal regions
Explain the interactions between distal-less and ultrabithorax in forming legs in Drosophila
Distal-less (Dlx) specifies leg precursor cells in the fly embryo and is expressed in the abdominal and thoracic segments. However, ultrabithorax (Ubx) is also expressed in the abdomen where is represses Dlx expression. Thus, legs form only in the thorax of the fly
How do Dlx and Ubx act differently in Crustacea to fruit flies
In crustaceans Dlx and Ubx are both expressed in the abdomen and thorax. However, Crustacean Ubx has an anti-repression motif that was lost in insects. Thus dlx expression is not repressed in Crustaceans and abdominal legs develop. These help crustaceans to swim
It was found that the Ubx protein in flies lacked an anti-repression domain in the ubx protein. What was seen when comparing the two copies of the ubx gene
There was a dramatic change in the Ubx C-terminus in flies. Crustaceans have a motif that block repression. On the other hand, Drosophila contain a polyalanine amino acid sequence which is absent in Crustaceans
What would be the results of ectopic expression of Crustacean ubx in the abdominal segments of Drosophila embryos
Limb formation in the abdomen due to loss of inhibition of dlx expression
What is meant by the term signal transduction
The process by which extracellular signalling molecules cause changes in target cells
List some of the downstream effects of signalling mechanisms on the cell
Survival, death(apoptosis), migration, differentiation, division
Signalling pathways usually involve an extracellular signalling molecule and a receptor protein that leads to the activation of intracellular signalling. Give examples of the different downstream targets of intracellular signalling
Metabolic enzymes, gene regulatory proteins and cytoskeletal protein elements
What type of signalling molecules do cell surface receptors often interact with
Hydrophilic ligands
What kind of signalling molecules do intracellular receptors interact with
Hydrophobic/lipophilic signalling molecules
What kind of proteins are often the downstream targets of intracellular receptors
Transcription factors
What is the other name given to ligand-gated ion channels
Inotropic receptors
Give an overview of signalling from GPCRs
The GPCR will interact with a ligand that allows binding of the receptor to a G-protein forming a complex which can then interact with enzymes
How many transmembrane domains are indicative of GPCRs
7 transmembrane domains
Describe the general structure and activation of enzyme-coupled receptors
Enzyme coupled receptors are transmembrane proteins that interact with ligands. This interaction then activates the intracellular enzyme domain or recruits an intracellular enzyme leading to signal transduction
Juxtacrine signalling is an example of a short-range signalling mechanism. Outline how this signalling mechanism acts
In juxtacrine signalling, two cells are in direct contact and the ligand is a membrane-bound signal molecule (transmembrane protein)
Which kind of short-range signalling involves ligand secretion from the signalling cell which acts as a local mediator on neighbouring cells, diffusing only a few cell diameters away
Paracrine
Describe what is meant by autocrine signalling
Autocrine signalling involves local groups of neighbouring cells with both produce the receptor and the ligand for a particular pathway. The secreted factor activates its own receptor on the same cell and neighbouring cells often as part of a positive feedback loop.
Where in the body is autocrine signalling often used
Used a lot in the immune system
In addition to paracrine, autocrine and juxtacrine signalling, there is another type of short range cell signalling. What is this signalling mechanism and how does it act
Short range signalling can also occur through gap junctions. Gap junctions act to physically connect cells together and allows them to share small molecules and ions. This acts to metabolically and electrically couple cells together and allows the transfer of species such as cAMP and Ca2+ between cells.
Cells of different types can respond to the same signal quite differently. Give an example of this
The response of different cells to acetylcholine can vary in different tissues. In the heart, release of acetylcholine causes the relaxation of cardiac muscle whereas in skeletal muscle it causes contraction. Similarly, in glandular tissue, acetylcholine release causes secretion of vesicles
What is meant by primary transduction
Primary transduction is the process the converts the signal from the extracellular signalling molecule to an intracellular signal
Intracellular amplification of signals is a common feature of signal transduction, T or F
T
Integration steps often occur in intracellular signalling where different pathways can affect the same components. In contrast, spreading of the signal is never seen, T or F
F – spreading is also seen
What is the role of scaffold proteins in intracellular signalling
Scaffold proteins play an important role in anchoring numerous components in place to respond to the activated receptors. By holding components in place, scaffold proteins effectively increase the effective concentration
What is often the result of phosphorylation of receptors once they become activated
Phosphorylation of receptors often creates recognition (docking) sites for downstream protein binding
What are the two main types of GTP binding proteins involved in intracellular signalling
Guanine Exchange Factors (GEFs) and GTPase activating proteins (GAPs)
What class of receptors are the receptor tyrosine kinases
Enzyme-linked receptors
What sorts of cell behaviours are RTKs involved in regulating
Proliferation, differentiation and migration
How many different families of RTKs are there
16 different families
Each ligand receptor pair involves one specific ligand and one unique receptor, T or F
F – whilst some ligands are specific for one receptor and vice-versa, some ligands and receptors can be promiscuous and bind to various other components
Give some examples of RTK ligands
Ephrins, Nerve Growth Factor, Fibroblast Growth Factor, Epidermal Growth Factor
What is the result of stimulating the EGF receptor tyrosine kinase
Stimulation of proliferation
What is the result of stimulating the IGF receptor
Stimulation of carbohydrate utilisation and protein synthesis
What is the result of stimulation of the TrkA receptor by binding of NGF
Stimulation of survival and growth of some neurons
Most RTKs are monomers with one major exception, which receptor is this
The insulin receptor is an RTK which is present as a dimer
What can be said about the extracellular domains of RTKs throughout the family
The extracellular domains vary greatly along with the ligands. They do however share features such as Ig-like and fibronectin-like domains and often contain several repeating units
Describe the structure of the intracellular domain of RTKs
The intracellular domains possess the kinase activity. These are present as a single domain or split into two
What can be said about the transmembrane domain in RTKs
The transmembrane domain is said to lack structure and be very simple. It is short and string like, consisting of between 25 and 38 amino acid residues
Outline canonical RTK activation
Following ligand binding, either as a dimer or monomer, the monomeric RTK receptor will dimerise by recruitment of the other receptor monomer. Similarly, ligand binding may also reorientate existing receptor oligomers. Activation of the RTK causes a change in conformation of the receptor dimer. This starts with the extracellular and transmembrane domains and is then translated to the intracellular kinase domain. This change in conformation of the intracellular domain unmasks the tyrosine kinase domain and exposes important residues for this process. The activated receptor then undergoes auto and crossphosphorylation. This increases the activity of the kinase domains, stabilises the active state of the receptor and causes the kinase domain to phosphorylate other tyrosines in the receptor to create docking sites. These kinase domains are now able to phosphorylate target proteins that bind to the docking site to transduce the signal.
What are the effects of auto and cross-phosphorylation of the active RTK
Increased kinase domain activity, stabilisation of the receptor active state (ligand independent) and the creation of docking sites for target proteins
Explain the gain of function approach that can be used to investigate RTK signalling
Genetically engineer DNA to generate a gene encoding an RTK whose extracellular ligand binding domain has been replaced with a homodimerization domain. Expression of this gene in an organism at high levels by incorporation of a transgene will result in the production of an RTK capable of dimerising in the absence of ligand binding. This receptor tyrosine kinase will be activated independently of the ligand and known as constitutively active. By expressing this transgene at high levels there is no need to interfere with the other endogenous gene.
Explain the loss of function approach that can be used to investigate RTK signalling
Genetically engineer DNA to generate a gene encoding an RTK whose intracellular kinase domain is mutated. This will lead to a loss of kinase activity and thus no auto and crossphosphorylation. Hence the RTK will be unable to activate in response to ligand binding. This DNA can then be expressed at high levels to result in a dominant negative or antimorphic mutation whereby the mutant RTK will poison the endogenous receptor
SH2 domains that bind to phosphotyrosines in RTKs also recognise adjacent residues. What is the recognition sequence which they recognise
Phosphotyrosine-Glutamate-Glutamate-Isoleucine
What component of the extracellular matrix do RTK ligands often form complexes with
Heparan sulphate proteoglycans (HSPGs)
The FGF receptor-ligand complex can become activated in the absence of binding to components of the extracellular matrix, T or F
F – the receptor can only become activated when in a complex with HSPGs
HSPGs can be membrane tethered in two ways. Describe these
HSPGs can be tethered to the cell membranes either by a transmembrane domain within the proteoglycan backbone or through a lipid modification such as GPI anchors
HSPGs can also be entirely secreted, T or F
T
What species attached to the proteoglycan backbones can be sulphated to trigger ligand binding to the FGF receptor
Glycosaminoglycans
HSPGs are important extracellular modifiers of cell-cell signalling, what is their role in the extracellular environment
They are important in organising the extracellular matrix into basal lamina
Give some examples of HSPGs
Glypican, Syndecan and Perlecan
Describe the structure of HSPGs
Consist of a proteoglycan core with glycosaminoglycan side chains
Other than Heparan Sulphate, give some examples of sugar side chains present on proteoglycans
Aggrecan, Betaglycan, Decorin and Perlecan
Modification by sulphation of GAGs can provide a code which creates binding sites for specific proteins and sequences that carry information, T or F
T
The pattern of sulphation acts as a code allowing specific HSPGs to interact with specific proteins, T or F
T
Describe the effects of HSPGs on the gradients of secreted molecules
HSPGs control the steepness of a secreted molecule gradient and how far a growth factor can diffuse through the extracellular space
Explain the role of HSPGs in FGF signalling
FGF and its receptor forms a complex with heparan sulphate proteoglycans. HSPGs provide an extracellular scaffold for FGF and presents it to the receptor after it has oligomerised on the HSPG
Give some examples or proteins that bind to RTKs
GTPase-activating proteins (GAPs) that function in the Ras/MAP kinase pathway, phospholipase C-? (part of the inositol lipid pathway) and PI-3 kinases that act as regulatory subunits
Explain how activation of RTKs leads to signal transduction by the Ras pathway
Ras is a smallGTPase that is present in the membrane of the cell. The activated RTK contains phosphorylated tyrosine residues in its intracellular kinase domain that have occurred because of autophosphorylation. These phosphotyrosines are recognised by proteins that contain an SH2 domain. In the Ras pathway, this protein is Gbr2 which binds to the activated receptor by its SH2 domain. Gbr2 then recruits another protein to the complex called sos by its SH3 protein-protein interaction domain. Sos is a guanine nucleotide exchange factor (GEF) that is bound to the Ras GTPase. Binding of Gbr2 to sos couples the activated RTK to the inactive Ras. Sos also promotes dissociation of GDP from Ras which is displaced by GTP. Now that it’s bound to GTP Ras dissociates from sos and phosphorylates MAP-KKK to transduce the signal further.
How does the MAP kinase pathway rely the signal transduction further from activation of the Ras GTPase
Activated Ras phosphorylates MAP-KKK which then binds to and activates MAP-KK by phosphorylation. Activated MAP-KK then goes onto phosphorylate and activate MAP-K. Activated MAP-Kinase can then phosphorylate transcription factors and other proteins leading to the regulation of gene transcription
What are the mammalian homologues of MAP-KKK, MAK-KK and MAP-K
MAP-KKK –> Raf, MAP-KK –> Mek and MAP-K –> Erk
What is the effect of having multiple stages in the MAP Kinase pathway
One activated MAP-KKK can phosphorylate and activate several MAP-KK proteins which in turn can phosphorylate and activated multiple MAP-K proteins. This acts as an amplification step in signal transduction
Cyclins are an example of downstream targets of MAP-Kinases, T or F
T
MAP-K is regulated by its phosphorylation by MAP-KK, describe how MAP-K is activated
In order to be activated MAP-K must have both of its phosphorylation sites on threonine and tyrosine residues phosphorylated by MAP-KK. These amino acids are interspaced by only one residue and lie in close proximity.
How long after RTK activation is gene transcription influence
Within minutes
Explain how Ras acts as a molecular switch in downstream RTK signalling
Ras is a smallGTPase that functions as a molecular switch. Its nucleotide-binding site is formed by several protein loops that cluster one end of the protein. Inactive Ras is bound tightly to GDP which is displaced by GTP when Ras becomes active. Ras can toggle between two conformational states depending on whether GTP or GDP is bound. The switch 1 and switch 2 regions change conformation dramatically between to two Ras states and this conformational change allows other proteins to distinguish activate Ras from inactive Ras. Active Ras binds to and activates, downstream target proteins in the cell signalling pathways. Hydrolysis of GTP to inactivate Ras requires the action of Ras-GAP which binds tightly to Ras burying the bound GTP. Ras-GAP inserts an arginine side chain directly into the active site. This Inserted arginine with threonine and glutamine side chains of Ras itself, promotes the hydrolysis of GTP.
Explain how the apparatus for fluorescent microscopy works
In addition to the light on the specimen, fluorescent microscopes also have a light of a specific wavelength shone onto the stage. This is either provided by a mercury lamp or a laser. The excitation filter of the microscope is then specific for the fluorophore used to stain particular regions of the specimen. A dichroic beam splitter mirror is used to reflect only short wavelengths hence allowing longer wavelengths to pass straight through. As fluorescent objects emit light of a longer wavelength than was shone onto it, the emitted light from the specimen passes through into the eyepiece. A greyscale camera then measures the light intensity and creates a greyscale image which can later be analysed
Why is a greyscale camera used to study fluorescence
Colour cameras aren’t sensitive enough to produce high quality images. Instead, colour is added to the greyscale image later to create what is known as pseudocolour
Explain how fluorescent resonance energy transfer can be used to investigate protein interactions
Recombinant fusion of proteins X and Y to separate fluorescent proteins that absorb and emit certain wavelengths of light allows you to determine if X and Y interact/bind. By correlating the wavelength emitted by the fluorescent protein attached to X with the wavelength of light needed for fluorescence of protein Y you can activate protein Y fluorescence if it is in close proximity to X (i.e. it is bound). I.e. if shining light needed for fluorescence in protein X leads to the appearance of light that is given off as a result of protein Y fluorescent you can determine that X and Y interact
What phenomenon is FRET said to rely on
Paired fluorescence
Leprechaunism is a disease caused by mutations in the insulin pathway. Describe the symptoms of this condition
Usually fatal within the first 2 years of life. Patients have an elfin-like facial appearance with protuberant ears and relatively large hands and feet. Also there is a decreased amount of subcutaneous fat and muscle mass is seen, and the skin is abnormal with increased hair growth
Where is insulin produced
In the B-cells of the Islets of Langerhans in the pancreas
Explain the cleavage events that take place after insulin synthesis
The full-length insulin protein is heavily modified after synthesis. These multiple cleavage steps leave the final product with only amino and carboxyl terminal peptides. These fragments are held together by cysteine bonds that can only form outside the cell
The cysteine bonding that holds the insulin peptides together can only form outside the cell, why is this
These cysteine bonds can only form in an oxidative extracellular environment
What is the immediate effect of insulin signalling
Glucose uptake from the blood into muscle cells and adipocytes
What is the long-term effects of insulin exposure
Increased expression of liver enzymes that synthesise glycogen as well as enzymes involved in triacylglycerol synthesis in the adipocytes
Describe the structure of the insulin receptor
The insulin receptor is also synthesised as a full length protein that is then cleaved into ? and ? subunits that are held together by disulphide bridges
The insulin receptor is an RTK, what is unusual about its structure
The insulin receptors is present as a dimer in its inactivated state
Describe what happens following ligand binding to the insulin receptor
Ligand binding brings the intracellular kinase domain together and results in autophosphorylation of the receptor. Autophosphorlyation creates a single docking site for the insulin receptor substrate (IRS)
Explain the role of IRS in insulin signalling
The insulin receptor substrate contains a phosphotyrosine binding domain (PBD) similar to SH2 that binds to the phosphorylated site on the activated insulin receptor. IRS then acts as a docking site for other proteins such as GRB2 leading to the activation of the Ras pathway.
