PA20293 gene transcription and DNA Flashcards
What determines expression rate?
How often genes get transcribed into proteins
What is the difference between genetics and genomics?
Genomics is the study of all the genes in an organism, how genes and proteins work together and interact.
Genetics is the study of inheritance of genes, mutations and inherited diseases.
Are all Polymorphisms harmful?
No, some are harmless, and are found in the non coding regions of genes (introns)
Polymorphisms are changes in gene sequence.
What does polygenic mean?
A disease caused by more than one gene being mutated.
These are very common
An example of a disease that isn’t polygenic ie is caused by a single gene mutation: cystic fibrosis.
What is an autosomal disease?
The gene causing the disease is not present on the sex chromosome.
Ie not X linked (XX, XY)
Autosomal dominant and autosomal recessive
Two copies of each gene are inherited by a child, one maternal one paternal. If a mutation occurs, when will a disease show up in a child? Think about dominant and recessive genes…
If the allele is dominant, only one copy of the mutated gene needs to be inherited. The disease will show up if the child inherits one mutated copy from one parent and it’s on a dominant gene.
If the child inherits two copies of a recessive mutated gene, one from each parent, the disease will show up. For a recessive linked disease to show up both copies need to be mutated.
What happens if a disease is X-linked, and in a male, there is a mutation on the X chromosome?
They will express the disease, males don’t have another copy to back it up as their chromosome is XY unlike females which are XX.
What happens if a disease is X linked, and In a female a gene is mutated on one of her X chromosomes?
She won’t express the disease as she has an back up healthy copy of the gene on her other X chromosome. She will be a carrier of the disease however. Females: XX
Does the sex of the child determine whether they inherit a disease or not with autosomal dominant/ recessive diseases?
No!
Only with X linked inheritance pathways
If a disease is autosomal recessive, and a child inherits one dominant copy and one mutated recessive copy from their parents, will they express the disease in their phenotype?
No,
They will be a carrier
For a recessive linked disease to show in the phenotype, 2 recessive genes must be present; homozygous recessive
Examples of diseases caused by X linked alleles?
Heamophilia and colour blindness
X linked diseases most often in males, most often X linked recessive
What are the three most common modes of inheritance?
Autosomal dominant, autosomal recessive, X linked recessive.
The 4th mode is X linked dominant but this is rare
What are molecular markers of disease?
Identifying molecular markers located along DNA which are inherited with disease, can help to locate the region of the gene causing the disease.
Molecular markers; map disease loci, they’re based on Polymorphisms
Polymorphisms act as convenient markers for studying inheritance
If you’ve inherited a polymorphism it’s likely you’ve inherited the genes around it
What are RFLP’s and what are they used for?
Restriction fragment length Polymorphisms.
Changes in sequences of restriction site of enzymes
Enzymes won’t cut at usual places,
Results in different sized fragments to usual
Inheritance of a polymorphism could indicate the likely presence of a linked allele for the inherited disease.
What are SSRs?
Simple sequence repeats
Repeat of 2-8 bases
Due to slippage of template
Can map disease loci, Map what’s maternal and paternally inherited
These random repeated sequences make an individuals gene profile unique
You’re looking for Polymorphisms and repeats when studying loci
Why do we want to map out/ locate genes that cause disease?
If we find out what the gene is we can target it for therapy, we can get an idea of what protein is involved, we can use the protein as a molecular target for treatment
What is gene mapping?
The process of determining the locus for a particular biological trait (phenotype) eg a disease.
Locus/ loci: the specific location of a gene / DNA sequence on a chromosome
Individuals get mapped at several different loci ie you’re usually looking at about 14 different loci: and you’re looking for different Polymorphisms and repeats.
Gene mapping:
Links are made between inheriting particular Polymorphisms and whether a disease is present or not.
You’re looking for mutations in genes near to inherited markers, these markers may be Polymorphisms, SSRs, RFLPs
What’s the problem with gene mapping?
It doesn’t narrow the search for our gene down that much, only to about 10 genes. We need to use techniques such as northern blotting to search for our effected gene more thoroughly.
Also gene mapping has no success with polygenic diseases.
True or false: a loss of heterozygosity can lead to cancer
True
Polymorphic, heterozygous regions may be lost where it results in the loss of a functional tumour suppressor gene. If the person suffers a point mutation in the back up gene copy too a loss of heterozygosity has occurred.
What is the concept of heterozygosity of genes?
The fact we have “back up” copies of genes in case a mutation occurs
When genomic copies from each parent have different bases in polymorphic regions due to SNPs, the region is heterozygous
You loose heterozygosity if there is a mutation in both a gene and the back up
What is contact inhibition?
Receptors on a cell surface touch another cell surface ligand, which results in cell growth inhibition, ie this is a stop signal. This is lost in cancer cells
True or false: It only takes one mutated gene to cause cancer
False.
Multiple changes in DNA are needed to convert a normal cell into a cancer tumour cell.
usually at least 3 diff genes need to be mutated
Eg Loss of : apoptosis pathway, kinase pathway and repair mechanisms.
What do single base mutations and sequence mutations lead to?
Single base: one different amino acid; could lead to loss of protein function but not definate.
Sequence: deletions/ mutations may scramble encoded mRNA: leads to complete loss of function protein.
Cancer requires 3-7 hits on relevant genes for it to occur. What does this mean?
3-7 mutations need to occur on relevant genes which are those regulating cell growth and division, DNA repair, and cell death.
Proto oncogenes are the mutated, cancerous form of oncogenes. True or false?
False. Oncogenes are the mutated form of the normal cellular genes Proto-oncogenes
This mutation is usually dominant
Receptors on cell surfaces receive growth signals. What can increase numbers of receptors/ increase their activity?
If more receptors are made; due to gene number being amplified, or translocation of receptors gene to an area of high transcription rate. Confrontational change in receptor; the receptor is always activated even without a Ligand Constitutive dimerisation (dimerisation does not need a signalling molecule)
In breast cancer,what does the her 2 receptor suffer, and what does this lead to?
Oncogenic mutations
Results in neu oncoprotein
Ligand independent, therefore constitutively active
In cancer, what does the EGF receptor suffer and what does this lead to?
A deletion
Leads to the Erb B oncoprotein
This is a constitutively active protein tyrosine kinase
Ligand independent
What is Li Fraumeni syndrome?
Inherited mutation in p53 gene.
It results in many childhood tumours
A mutation in the ____ tumour suppressor gene leads to a retinoblastoma
pRB
Mutation in the second allele leads to retinoblastoma, a tumour in the eye
What are translocations?
Abnormal crossovers between chromosomes during cell division
They cause a split in a gene, and joining with another part of a gene
Can lead to genes normally regulated by one promoter being separated from this promoter region and instead being controlled by another promoter.
How do chronic myeloid Leukaemias occur?
Translocations: result In constituitively active c-abl tyrosine kinase,
by loss of the N-terminal regulatory domain (due to this translocation, lost part of gene therefore lost part of protein)
Leads to increased cell signalling and growth: forms tumour
Does each gene have the same upstream sequence?
NO
Each gene has a different upstream sequence
Different transcritpion factors therefore bind to different upstream elements, and specificity arises from these different transcription factors used. Different transcription factors switched on results in switching on of different genes.
Which Direction does RNA polymerase work in?
From 5’ end to 3’ end of DNA
Moves away from promoter region toward the downstream part of the gene
(but reads in 3’ to 5’ direction)
What assembles at the TATA box? What does this then recruit?
The initiation complex assembles here
This is where transcription factors come together an bind to the TATA box and then recruit RNA polymerase.
What does the position of the TATA box (~25 base pairs from start site) enable?
This is where RNA polymerase will bind, so the distance of the TATA box to the start of the gene will be correct in order to align the front of RNA polymerase enzyme up with the First base of the gene to be read!
The TATA box is CRITICAL in the positioning of RNA polymerase, and therefore where transcription starts from
What do RNA polymerase I, II & III translate DNA into?
RNA pol I: into rRNA, in nucleolus
RNA pol II: into mRNA, in nucleoplasm
RNA pol III: into tRNA, in nucleoplasm
What must all amino acid sequences start with to make a functional protein?
Must start with the start codon, methionine (Met)
How is the mRNA strand positioned when it attaches to the ribosome?
mRNA is sandwiched between the two subunits of the ribosome (the large and small subunit)
tRNA molecules bring in amino acids that match the sequence on the mRNA strand
What’s the regulatory region of DNA called?
The promoter region
Contains enhancer region, upstream elements, TATA box where RNA polymerase binds and start site.
These are several highly conserved regions bound to by regulatory proteins.
What is gene transcription mediated by?
A transcription complex which includes RNA polymerase II, this is the initiation transcription complex that assembles at the TATA box and then proceeds to move along the gene
What position is the start site of a gene? What is everything to the left (upstream ) of this?
Start site is position +1
Everything to the left upstream of This is negative numbers, everything to the right downstream is positive numbers
What do the enhancer region and upstream elements bind?
These bind constitutuive and inducible transcription factors
What are constitutive transcription factors?
There all the time, present in all cells all the time!
What are the inducible transcription factors? When are they activated?
These are regulated downstream of receptors and will only get activated when the receptor is activated and therefore the signal pathway is activated.
These TFs aren’t present all the time!!
What are upstream elements also called?
Promoter-proximal elements
(these are any regulatory sequence located within ~200 BP upstream of the start site
Where is the enhancer located? Upstream or downstream? Is it one enhancer per gene?
It can be located upstream or downstream of the gene
One enhancer can oversea several related genes, it’s not just one enhancer per one gene
Are transcription factors part of RNA polymerase?
No, RNA pol is a separate enzyme, the only thing they have to do with RNA pol is that basal/ general TFs associate and then recruit RNA pol to form the transcription initiation complex, which allows transcription to occur.
General/ basal TFs are coassociated with RNA Polymerase
What are the TF-11 family coassociated with?
RNA pol II
During the formation of the preinitiation transcription complex, where do the TFs first bind?
To the TATA box
Eg in preinitiation complex involved RNA Pol II, TF11D first binds to the TATA box with its TBP (TATA binding protein).
During the transcription initiation complex (preinitiation complex) formation, what distorts the structure of DNA?
TBP binds to the major groove of the TATA box of DNA.
TBP is a protein, if you stick a protein onto coiled DNA it will bend it and distort it. TBP binding results in considerable bending of the DNA. ☡
Overall, what does the formation of the transcription Initiation complex involve?
It’s a slow build up of associated proteins (transcription factors)
Emphasis that this is a STEPWISE assembly of TFs coming together.
The transcription initiation complex comprises associated general transcription factors and RNA Pol II
Which transcription factor activates the whole transcription initiation complex at the end?
TFIIH, it’s the final protein to bind.
It has helicase activity, so can unwind the DNA strand for the gene we need.
It also has kinase activity, and phosphorylates the carboxyl terminal domain of RNA Pol II
Once the RNA polymerase starts moving downstream, all the basal/ general TFs dissociate and are released, all except from ___?
TBP
TBP, TATA binding protein, stays bound
When the transcription initiation complex is first formed, where does the active site of RNA pol II lie?
Above the start site of the gene, the TATA box is located in a position so that it is the correct distance away to allow the front of RNA pol to be aligned with the start of the gene!
What kind of histones does euchromatin contain?
Acetylated histones
Relaxed DNA binding around chromosomes, actively transcribed DNA
What is a nucleosome?
A basic structural unit, DNA is wound around a protein called a histone.
Nucleosomes are the core OCTOMER of histone proteins
146 BPs of DNA wound round each histone core, 2 histones in each OCTOMER
DNA is very tightly packed (wrapped) around these histones
What’s the name of the structure whereby DNA is coiled really tightly so it’s tightly packed? Diameter?
DNA coiled into SOLENOID structure
30 nm diameter
