GENE 7: Expressing the genome Flashcards
What is gene expression?
The process by which the information from a gene is used to synthesise a functional gene product, which is either a protein (if the gene is a protein coding gene) or a functional RNA (if it is a non-protein coding gene).
from 5’ to 3’ list the order of the structure of a protein coding gene
5’ - Upstream enhancers > Promoter > TATA box > %’ UTR > Exon 1 > Intron 1 > exon 2 > Intron 2 > exon 3 > 3’ UTR - 3’
What are the three stages of gene expression?
Initiation, elongation and termination
Where do basal transcription machinery assemble on the DNA during initiation?
The promoter
What are the three common features of promoters for protein-coding genes?
- start site
- TATA box
- sequences bound by transcriptional regulators
What does the basal transcription machinery comprise of?
RNA polymerase II
5 multi-subunit general transcription factors
What are the 5 multi-subunit general transcription factors called?
TFIIB, D, E, F and H
Boris Didn’t Eat French Ham
TFIID comprises the TAT-binding protein (TBP) and around ___ TAFs (TBP-associated factors)
11
What is the first component to bind to the promoter?
TFIID
What forms the initiation complex factors?
TFIIB, E, F, H and RNA polymerase II
Describe the processing of primary RNA transcripts
co-transcriptionally
5’ capping as soon as transcription has been initiated
splicing and editing while the transcript is still being made
3’-polyadenylation as an inherent part of termination mechanism for RNA polymerase
What is 5’-capping?
The addition of 7-methylguanine
Define transcription factor
Sequence-specific DNA binding proteins that bind at or close to the core promoter and influence the efficiency of transcription initiation
Define DNA helicase
A subunit of TFIIH that uses energy from the hydrolysis of ATP to open up the DNA double helix; allowing RNA polymerase II to have access to the template strand
Define transcriptome
The total complement of mRNA molecules (or transcripts) produced in a specific cell or the population of cells comprising a tissue
Name three methods of measuring gene expression
- qPCR
- gene expression microarrays
- RNA-Seq (RNA sequencing)
What can qPCR do?
Can quantify a specific transcript whose cDNA primers have been designed to amplify
What can gene expression microarrays do?
can simultaneously detect and quantify transcripts for thousands of genes within a particular mRNA sample. Fluorescently labelled RNA hybridises to transcript-specific oligonucleotides arrayed on a solid support.
What happens in RNA-Seq (RNA sequencing)?
is a ‘next generation’ technique where a cDNA library is made and sequenced. The sequences tells us which genes are expressed within a sample and the number of reads indicates expression levels.
When does RNA polymerase II bind to the TATA box?
Only after TFIID, A, B have bound to the TATA box. RNA pol II will only bind after and when attached to TFIIF, followed by further transcription factors
What information is required for qPCR?
Oligonucleotide PCR primers based on known transcript sequences
When studying the transcriptome using RNA-Seq, why is each transcript’s exact RNA sequence not needed to be known?
While the genome sequence is required to identify which gene each read maps to, RNA-seq reads can pinpoint novel splice junctions and fusion genes.
What is the most common DNA modification in mammals?
DNA methylation
Where does DNA methylation occur? What does this produce?
5th carbon of cytosine ring, produces 5-methylcytosine.
CpG sites
5’-C-phsophate-G-3’
What does DNA methylation do to the structure of DNA? What does this lead to?
Distort the DNA helix, which inhibits transcription
How many CpG islands are there in the human genome?
~30,000 (aprox. 15.% of human genome)
What does CpG islands mean?
stretches of 0.5-2 kb of DNA with a greater frequency of CpG dinucleotides than the rest of the genome
When a CpG island in the promoter region of a gene is methylated, what happens to the expression of the gene?
It is usually silenced
Many CGIs occur at ____ _____
gene promoters
What is genomic imprinting?
A form of epigenetic inheritance, where DNA methylation ensures only one parental allele is expressed. When the paternal allele is expressed, the maternal copy is silenced and vice versa.
How does Prader-Willi Syndrome (PWS) come about?
Genetic imprinting disorder: which is often caused by deletion of the paternal allele in the region on chromosome 15 containing the gene SNRPN. This occurs after the maternal allele has already been silenced by imprinting, leaving no expression of SNRPN.
What happens to PWS patients?
They suffer from extreme feeding problems, including hyperphagia, or extreme, insatiable appetite and obsession with food. Affected children are also developmentally delayed for motor skills due to decreased muscle tone.
What happens in Angelman syndrome?
Loss of expression in a region of chromosome 15. In this case it is usually caused by loss of the maternal allele when the paternal allele has been silenced by imprinting. Loss of the gene UBE3A results in disorder of the nervous system characterised by developmental disabilities, seizures, speech deficits, and motor oddities.
In addition to DNA base modifications, what else is used to regulate gene expression?
Post-translational modifications of histones proteins. These can influence the activity status of the chromatin
Which modifications of Histone H3 promote gene transcription?
- Acetylation of Lysine 27
- Tri-methylation of histone H3 lysine 4 results in active promoters and gene expression
