Theme 3 Flashcards
promoters
regions of a few hundred base pairs where RNA polymerase and associated proteins bind to the DNA for transcription
TATA box
5’-TATAAA-3’
a usual eukaryotic promoter region on the DNA to be transcribed
terminator
transcription continues until RNA polymerase reaches a signal on the DNA that signals transcription to stop
housekeeping genes
DNA that contains genes required for all normal functions of the cell
- they are expressed constitutively, always transcribed and translated bc they allow for constant maintenance of general cellular activities
ex. structural and ribosomal proteins
regulated genes
are turned off and on as needed
- bring about changes that can result in growth/divisions
ex. enzymes
sigma factor
mediates promoter activity in bacteria by associating with RNA polymerase as it facilitates binding to specific promoters
why is gene regulation important?
because environments can undergo changes and cells need to be able to adapt to these changes
preferred nutrition source of E. coli
why the plateau?
glucose, when glucose is gone, bacteria pop growth plateaus and then growth begins again on lactose until it runs out
- plateau bc it’d be a waste of resources to metabolize lactose synthesizing enzymes when there’s no lactose present
beta galactosidase
responsible for breaking down lactose in glucose and galactose
- produced by turning on beta-galactosidase gene and is only done when glucose is gone and lactose is available
amt of B-galactosidase production depends on what?
the amt of B-galactosidase produced increases in response to the addition of lactose to the medium
- it depends on the presence of lactose
what is gene expression
it includes all the steps of a protein being made, modified and regulated
- transcriptional control from DNA to mRNA
- translational control from mRNA to protein
- post-translational control from protein to an activated protein
- if any of these steps are disrupted then there is no activated protein
transcriptional control of gene expression
when proteins bind to the promoter region, it increases the binding of RNA polymerase
- so by controlling the binding of these proteins the cell can activate or inhibit transcription
translational control of gene expression
the ribosome binds to a specific region on mRNA in prokaryotes and eukaryotes
- speed of translation depends on stability of mRNA (how quickly degraded it is)
post-translational control of gene expression
allows an inactive polypeptide to fold into functional 3-D protein
- whether these modifications occur or not depends on whether the protein gets activated
which component of gene expression regulation is the fastest?
post translational modifications are
- they allow a cell to have a stockpile of protein in the cell that inactive so that when the cell receives a certain signal, it can just modify the proteins to activate them; the response can be brought on by quick changes to the env’t
which component of gene expression regulation is the slowest?
transcriptional regulation is the slowest
- the cell starts from scratch and the regulation here is a result of more drastic environmental changes that the cell is exposed to for a longer amt of time (ex. converting from glucose metabolism to lactose metabolism)
which component of gene expression regulation is the most efficient?
transcriptional regulation is the most efficient
- cell doesn’t waste any energy/resources making mRNA or protein unless it needs it
ex. E. coli only starts transcribing beta-galactosidase when lactose is present
components of a bacterial operon
- promoter
- operator
- coordinated gene cluster
what has to happen for lactose metabolizing proteins to be expressed?
- glucose is depleted
- lactose is present
lactose permease
a transport protein that sits in bacterial cell membrane and allows the transport of lactose into bacterial cells
lacZ
the gene coding sequence for beta-galactosidase
lacY
the gene/coding sequence for the transmembrane protein lactose permease
structural genes
code for the sequence of amino acids making up the primary structure of each gene
ex. lacZ, lacY
operator
lacO in the lac operon
- binding site for the repressor
lacI
codes for a repressor protein in lac operon (controls expression of lacY and lacZ genes) because the repressor protein binds to the operator and inhibits transcription from occurring = negative transcriptional regulation
lacP
codes for a promoter whose function is to recruit RNA polymerase complex and initiate transcription
polycistronic mRNA
a single molecule of mRNA that formed by the transcription of functionally related genes located next to one another on a bacterial DNA
- here, each of the coding sequences is preceded by a ribosome binding site so translation can be initiated there
operon
region of DNA consisting of the promoter, operator and coding sequence for the structural genes
negative transcriptional regulation of lac operon
how does this work?
a regulatory protein binds to the DNA near the gene (operator) where transcription gets prevented
- once all 4 subunits bind to the lac operon DNA, the DNA is twisted in a loop and transcription can’t occur
positive transcriptional regulation of lac operon
a regulatory protein binds to RNA at a site near the gene in order for transcription to take place
- in the absence of glucose, positive regulation produces lactose permease and B-galactosidase caused by an increase in cAMP
allosteric inhibition on the lac operon
lactose allosterically inhibits the repressor protein when present
-lactose acts as an induced molecule by binding the repressor proteins and causing them to change shape so they can’t bind to the DNA
adenylyl cyclase
catalyzes the production of cAMP from ATP
- when glucose levels are high adenylyl cyclase activity is inhibited, when glucose levels are low, the activity is high and more cAMP is produced
what does the concentration of glucose have to do with the amount of cAMP (lac operon)
they are inversely related
- high glucose conc means less adenylyl cyclase activity and less cAMP
- low glucose means adenylyl cyclase is active and more cAMP is produced
CRP
and what is has to do with positive regulation of the lac operon
cyclicAMP receptor protein (or catabolite activator protein)
- when cAMP is bound, CRP binds to a different site on the DNA called the CRP-cAMP binding site
- thus the amount of cAMP present determines whether an activator will bind to this site and cause transcription or not
allosteric activator in the lac operon
when cAMP is present in high levels, it can bind to CRP as an allosteric activator, inducing a change of shape in the transcriptional activator protein and allows it to bind to the DNA
when are levels of lac operon mRNA the highest?
lowest? when is there none at all?
highest: at the end of the curve after growth on lactose
lowest: when glucose is all used up, RNA polymerase will just start transcribing mRNA for the enzymes needed to metabolize lactose
none at all: at the beginning of the E. coli growth curve because bacteria will be actively using glucose
CRP-cAMP complex
a positive regulator of the lactose operon
- cAMP is an allosteric activator of CRP binding to DNA
about how many different types of cells do adult humans have?
about 200 types which all differentiate from a single zygote
- even though all cells have the same genome, gene regulation differences lead to different proteomes that have diff. cellular functions
transcription factors
proteins that bind to specific DNA sequences in eukaryotes
- control transcription of DNA to RNA which contributes to gene regulation
- often work w proteins that can change different things in cell division and dev’t
how does the way the DNA is packaged in eukaryotes have an impact on whether the genes get transcribed or not?
when DNA is coiled (chromatin), it’s not accessible to transcription proteins, chromatin remodelling must occur to allow transcription to happen
chromatin remodelling
when chromatin must loosen from histone proteins for transcriptional machinery to work
- nucleosomes are repositions to expose diff stretches of DNA to the env’t
== unwinding of DNA from histones
how is chromatin remodelled?
chemical modification of histones
- addition or removal of methyl and acetyl groups to the histone tails (strings of AAs that protrude from histone proteins)
- usually methylation or acetylation occurs on lysine residues of histone tails
what happens when you add a methyl group to the base of a cytosine on a histone protein?
- chromatin structure changes and and transcription is restricted
- this produces a CpG island (bc methylation happens on C nucleotides base paired w Gs)
CpG islands
clusters of CG nucleotides located near the promoter of a gene
- heavy methylation of cytosine leads to repression of transcription near the island because it prevents binding of RNA polymerase
epigenetic
mechanisms of gene regulation that involve changes to the way that the DNA is packaged, rather than to the DNA itself
imprinting
the sex-specific silencing of gene expression
ex. an allele inherited from the mother is repressed and so the allele from the father is the only one expressed
transcriptional activator protein
in eukaryotes, they bind to enhancers
- they help control transcription of genes in different cells will occur
enhancer sequence
in eukaryotes
a DNA sequence that transcriptional activator protein binds to, which allows transcription to begin
mediator complex of proteins
in eukaryotes
- once transcriptional activator protein has bound to the enhancer sequence this mediator complex recruits RNA polymerase complex to bind to the promoter
adenylation and addition of 1 methyl group results in
transcriptional activation
the addition of 3 methyl groups results in
repression of transcription
what are the ways that DNA binds with transcription factors
basic helix-loop-helix
helix-turn-helix
zinc finger
leucine zipper
how do trans-acting factors interact with DNA
- most of the transcription factors have alpha-helical domains that fit nicely within major grooves of DNA
- these are generally the result of the interactions b/w AAs in alpha helix and functional groups of nitrogenous bases
TBP
TATA-box binding protein
- a subunit of the transcription factor TFIID
BRE region
B recognition element
- recognized by TFIIB
ok eukaryotic transcription
- general transcription factors bind to promoter, transcriptional activator proteins bind to enhancers
- looping of DNA results in lots of things coming into close quarters allowing transcription to occur (transcriptional activator proteins, mediator complex, RNA poly II, general transcription factors)
transcriptional repressors (eukaryotes)
can stop transcription when they bind to DNA sequences called silencing regions (loc upstream of where transcription complex is); when the region is activated, it leads to interference of general transcription factor assembly and mediator activity req’d for transcription
human globin gene expression and dev’t
a fetus has 2 alpha-globins and 2 gamma-globins: gives them a high affinity for oxygen
an adult as 2 alpha-globins and 2 beta-globins: have a moderate affinity for oxygen
– basically, fetuses have the beta globin gene turned off and gamma globin gene on while this is opposite for adults
– this is a result of chromatin being tightly wound where genes are turned off and being unwound where genes are on to allow transcription to occur
histone deacetylases
HDAC
promote removal of acetyl groups from other histones
- results in repression again of transcription when nucleosomes reassemble
- an example of some proteins that can only bind to methylated regions of the DNA
what is the main difference b/w transcription in prokaryotes and eukaryotes?
default state of transcription
- for prokaryotes it is on and in eukaryotes it is off
what does multi level gene regulation allow in terms of gene expression?
it allows the cell to rapidly alter the levels of active protein in response to internal and external signals
in situ hybridization
allows us to compare and study 1 or more genes of interest (generally a few)
how do DNA microarray chips work?
on a glass slide, tiny spots that contain known sequences of DNA act as probes to detect gene expression (aka transcriptosome)
- it allows is to compare thousands of genes at once
how do we use DNA microarrays?
ex. to analyze differences in gene expression
- we learn that not all genes are active at once
- we see which genes are active at certain times and see what changes occur when gene expression is altered
fluorescently labelled cDNA
complimentary DNA that has be created through reverse transcription with fluorescent nucleotides (from 2 diff sources) then combined in equal amounts and are added to microarray chip and we can fluorescently analyze the gene activity to see which genes are expressed under certain conditions (ex. normal vs carcinogenic)
- more active genes are more fluorescent
mRNA stability
in order to stop gene expression, mRNA gets degraded by siRNAs and miRNAs: some mRNAs get transcribed but not translated
siRNAs
small interfering RNAs, lead to mRNA degradation
also assoc w RISC complex, are exact complements of mRNA target and bind to sequence by association
- additionally induce cutting of mRNA and destabilizes target mRNA even more
miRNAs
microRNA, lead to inhibition of translation
form hairpin loops (bc of base-pairing w/in miRNA) and base pair (bind in a NONexact manner) with some mRNA that needs to be regulated, allowing RISC complex proteins to inhibit translation
RISC complex
RNA induced silencing complex
small, single stranded RNA target RISC complex to specific RNA molecules by base-pairing regions with the target and depending on the type of small RNA, will inhibit mRNA translation in some way
proteasomes
large protein complexes that can break peptide bones and degrade unneeded/damaged proteins
- post-translational modifications allow cells to activate or deactivate certain proteins
- ATP dependent, recognize ubiquitin on proteins and break down
selective degradation
limits the length of time by which a protein can function in a cell
ubiquitin
a small protein that tags/identifies proteins (is added covalently) to be degraded by proteasomes
