Chapter 1 : Genomics and Regulation in Eukaryotes Flashcards
What is the genome?
organisms complete set of DNA, including chromosomal and mitochondrial DNA
Describe Genomics?
- study of the genomes of organisms, including determination of the entire DNA sequence of organisms and fine-scale genetic mapping
Describe the human genome project?
- international research effort to sequence and mapp all of the genes of the human genome
- used for biomedical studies
- used to look for genetic variation that increases risk of specific diseases such as cancer
- discover the genetic basis for health and disease
Compare the genomes of E.coli, nematode and homo sapien
- similar in number of protein encoding genes, large difference in amount of non-coding regions in the genome
Explain the surprising results of the post-genomic era
- number of protein encoding genes did not correlate with complexity as expected
- (humans substantially fewer protein-coding genes)
- prevalence of the use of other mechanisms that increase complexity and variation without increasing number of coding genes
- recognition of a complex regulatory network
- “junk DNA” serves a purpose
What is gene expression
- information from a gene is used in the synthesis of a functional gene product (often proteins)
- in non-protein coding genes (tRNA, snRNA),product is a functional RNA
What are some steps where regulation of gene expression can occur
- regulation of transcription
- RNA processing
- RNA transport and localization
- mRNA sequestration and degredation
- translation
- post-translational modifications
- protein degredation
What are the complex cellular networks involved in regulation of the human genomr
- differential gene expression
- combinatorial gene control and protein interaction networks
- complex signal transduction pathways
How can a single fertilized egg give rise to a complex organism with cells of varied phenotypes?
- all cells have same genetic info
- differences in cells result from differences in gene expression
- multicellular development, differences in gene expression are set up and maintained by epigenetic mechanisms
- epigenetic control provides for cell differentiation and perpetuation of expression states and cell memory
Epigenetics is….
the study of biological mechanisms that will switch genes on and off
- NOT a change in DNA sequence
What is an epigenetic mark?
modification of DNA or histones
What is included as an epigenetic mark?
- dna Methylation
- Histone modifications
What is an epigenetic pattern?
- patterns of DNA methylation and histone modifications
Are patterns heritable?
yes
What are some factors that may influence epigenetic patterns?
-
What are some factors that may influence epigenetic patterns?
- Diet
- geography
- sleep
-exercise patterns - aging
- environmental & behavioral factors
Are epigenetics reversible?
- yes
What is a non-coding RNA (ncRNA)
- An RNA molecule not translated into a protein
- it is possible that many ncRNAs are non functional, but they could also not be
What are the functions of regulatory RNAs?
- bind to DNA to block transcription of the target gene
- target specific mRNAs for destruction
- block translation of the mRNA
What are the two broad classes of regulatory RNAs?
- micro RNAs (microRNAs)
- Long noncoding RNAs (lncRNAs)
Describe early and late stage regulation?
- early stage: economical, but it takes time (only transcribes proteins that it actually needs)
- late stage: very fast, not economical
Do eukaryotic polymerases recognize their core promotor sequences?
no
What is the function of a trans-acting regulatory protein?
- bind cis-acting regulatory sequences to control eukaryotic transcription
Describe regulatory DNA sequences?
- cis-acting
- core promotor region
- proximal elements
- enhancer sequences
What are enhancer sequences?
- ## enable genes to be transcribed only when proper transcriptional activators are present
What are silencers?
- decrease in gene activity when bound to a TF
What are topologically associated domans?
(TADS) are relatively large sections of DNA
- enhancers and silences can act within these areas
- TAD boundaries are “insulators” which prevent enhancers and silences in one TAD from affecting transcription of genes in another
What are transcription factors?
- TFs are proteins that bind DNA regulatory regions to activate/stimulate or repress transcription
- trans-acting regulatory proteins
What is the general role of a transcription factor?
- bind to the promotor region
- required for RNA polymerase binding
What are two examples of a TF?
- activator
- repressor
How are TF’s grouped into families?
- DNA binding domains
Describe transcription activation.
- general TFs bind to the promotor region
- General TFs facilitate binding of RNA polymerase to the promotor (pre-initiation complex)
- activator TFs bind to proximal and enhancer sequences
- activators bound to the enhancer region work with mediator proteins to initiate transcription
Describe modulation of transcription
- activator proteins bound to DNA at an upstream enhancer
- attract proteins to promotor region
- these proteins activate RNA polymerase and thus transcription
- DNA can loop around on itself to cause interaction between activator proteins/ proteins that mediate RNA polymerase activity
What is a mediator
- large multiprotein complex, required for transcription by RNA polymerase II
- functions as a transcriptional coactivator
- regulates various steps transcriptional activation
- main function: transduce signals from enhancer-bound TFs to the components of the preinitiation complex
What is an enhanceosome
- higher-order protein complex assembled at the enhancer and regulates expression of a target gene
Humans have a low number or protein-coding genes. Where does our complexity come from?
- human complexity depends on combinatorial control of gene expression
- in other words, how those genes are regulated in used
What is the effect our complex system of multiple regulators?
- the same gene can be transcribed in multiple ways
- depends on combination, presence or absence of various transcriptional regulator proteins
What is combinatorial regulation?
- a specific combination of TFs are needed to turn the gene on
Describe the many results from an interplay between combinatorial regulation?
- different enhancer regions (modules) can be used by different TFs to activate same gene
- expression of different genes can be coordinated by a single TF
- different roles may be fulfilled by the same TF
- different combinations of a few TFs can generate many different results
- many TFs work together as a committee, combining their effects to determine final transcription rate
What are alternative promotors?
- promotors that are active and allow for independent regulation in different situations
What are 3 ways to identify and study regulatory sequences
- mutation analysis
- identify conserved non-coding sequences across species
- evaluate gene expression with reporter genes
How to evaluate gene expression with reporter genes?
- promotor and enhancer analysis
- regulation by micro RNAs and lincRNAs
- temportal and spatial expression
What is a mutation analysis?
- create a gene mutation and observe the consequences in order to uncover the function of a gene
- mutations that result in lower transcription indicate nucleotides that are important for transcription
What does it mean to identify conserved non-coding sequences across a species?
- align sequences to determine identities (look for similarities among the sequences)
How can you study the strength of a regulatory region when you cant easily assay them for the gene product?
- substitute the gene with a reporter gene, one that encodes a product that you can easily assay
What is the purpose of a reporter gene plasmid?
- sequences of interest are cloned into reporter gene plasmids
- contains reporter gene and other gene s necessary for plasmid maintainance/gene expression
Reporter plasmids may be used to study:
- promotor and enhancer analysis
- regulation by micro-RNAs and lincRNAs
- temporal and spatial expression
Regions cloned into the reporter plasmid/vector depend on whether one wants to study the:
- promotor regions 5’ to the gene of interest
- regulatory regions in the 3’ UTR
transcriptional fusions can be used to study:
- promotor and enhancer analysis for regulatory regions
- regulation by micro rnas and linc rnas
- spatial location: cell types, tissue types
- temporal location of gene expression
- subcellular localization
describe pair-rule genes
- pair-rule genes are expressed in alternate parasegments during development of segmented insects
ex: expression of the pair-rule genes even-skipped (eve) and fushi tarazu (ftz) in alternating bands in drosophilia early embryo… each band corresponds to one parasegment
What are the three examples of differential gene transcription?
- expression of beta-globin genes
- expression of Sonic Hedgehog
- regulation of yeast GAL regulon
Describe the expression of beta-globin genes (1st example of differential gene transcription)
- regulation in response to a developmental program
- six closely related globin genes form beta-globin complex
- each gene produces distinct beta-glovin polypeptide with unique oxygen-carrying capacity
What is a locus control region
- highly specialized enhancer
- regulates transcription of multiple genes packed into complexes of closely related genes
Describe the graph showing developmental expression of beta-globin-complex genes
- two beta-globin polypeptides join with two copies of alpha-globin to form hemoglobin
- at bird, hemoglobin molecule is made of 2 alpha chain and 2 gamma chains.
- gamma chains gradually replaced by. beta chains as infant grows
(gestation = gamma + alpha dominate)
(birth = beta dominate)
Describe expression of Sonic Hedghog (2nd example of differential gene transcription)
- regulation for tissue-specific expression
- due to the action of two different enhancers
- one combo of regulatory proteins binds brain enhancer in brain tissue, a different combo binds limb enhancer in developing limbs
Describe regulation of the the yeast GAL regulon? (3rd example of differential gene transcription)
- regulation in response to changing conditions
What is a regulon?
- group of genes regulated as a unit, controlled by same regulatory gene that expresses a protein acting as repressor/activator
Eukaryotic version of lactose?
Galactose
Cellular response to high [glucose]
- represses genes for alt C-source use (galactose operon)
- induce genes for efficient glucose use
Cellular response to low [glucose]
- represses genes for glucose uptake
- induces genes for gluconeogenesis, genes fot alt C-source use (galactose)
Describe GAL regulation
- defined by Gal4 which regulated over 22 galactose-related genes, using structural and regulatory GAL genes
What is GAL4
- DNA -binding transcription factor
- responds to galactose
- binds to the UAS of GAL genes activating the GAL genes (UAS = upstream activating sequence)
Describe transcriptional activation by GAL4 when GALACTOSE is ABSENT
- Gal4 recognizes 17 gp seq. (UAS)
- binds to UAS as a dimer
- inactive when bound to Gal80 (Gal80 acts as a repressor, cannot activate transcription
Describe transcriptional activation when GALACTOSE is PRESENT (glucose absent)
- Gal3 binds to galactose, then to Gal80
- bind of Gal3-Galactose to Gal80 induces conformational change in protein, Gal80 leaves from Gal4
- release of gal80 from gal4 = gal4 is active, transcription can activate
What is SAGA?
- recruited by an activated Gal4
- transcriptional co-activator complex
- histone modification
- allows chromatin to relax in order for the promotor to open
What are the sequences of events in the activaton of Gal4- regulated genes
- Gal 3 moves gal80 to cytoplasm, gal4 is now active
- gal4 recruits SAGA
- gal4 recruits mediator and SWI/SNF
- SWI/SNF along with SAGA remove nucleosomes from promotor
- SAGA and mediator recruit GTFs and RNA poly II, forms pre-initiaion complex
