Gene Regulation Flashcards
Why do Bacteria need to regulate Genes?
- they are nutritional opportunists
- to do so, they’ve evolved regulatory systems that couple expression of gene products to sensory systems that detect relevant compound in enviro.
Basics of Prokaryotic Transcription
- Promoter & Operator
- Activators & Repressors
- Positive & negative regulation
-Promoter: where RNA polymerase binds - allows transcription to occur
-DNA seq near promoter serve as binding sites for seq-specific regulatory proteins called activators & repressors
-activators physically help tether RNA poly to promoter
-repressors either physically interefere w/ binding or impede movement of RNA polymerase
Operator: where repressors bind
Positive Regulation: when activator protein must bind to its target DNA site as necessary prerequisite for transcription
-the PRESENCE of bound protein needed for transcript
Negative Regulation: when repressor protein must be prevented from binding to its target site for transcript to begin
-the ABSENCE of bound repressor allows it to begin
Gene Regulation; Bacteria
-how bact. genes organised
- Regulatory gene
- role of regulatory protein
- have related gene together in clusters (OPERONS) so that genes required for similar functions can be made together
- Regulatory gene (may be close or far from operon) makes regulatory protein
- regulatory protein can either be inhibitory or enhancing to transcript
Gene regulation; Prokaryotes
- 2 ways regulation achieved
- Sigma factors
2 ways: Sigma factors or regulatory proteins
SIGMA FACTORS:
-recognizes genes by their promoters (recognizes specific set of sequences at -10 & -35 regions
-Therefore, set of genes required for particular function can have same promoter regions & be activated by a given sigma factor
Gene regulation; Prokaryotes
- Regulatory proteins (2 types)
- trans & cis acting factors/repressors
Regulation by REGULATORY PROTEINS
-regulatory proteins bind to target site - can control expression
Positive regulator: Protein binds & transcription starts (called positive control)
-trans-acting factors must bind to cis-acting sites for RNA polymerase to initiate transcript. at promoter
Negative regulator: Protein binds & transcription stopped/inhibited (or binding suppresses expression) (called negative control)
-trans-acting repressor binds to cis-acting operator to turn off transcription
Gene regulation; Prokaryotes
-Induction and repression
Induction: process where gene expressed in response to presence of specific molecule called an inducer
Repression: When the gene is switched off in response to presence of specific molecule called repressor
Gene regulation; Prokaryotes
-MalT operon - e.g. of positive regulator and induction
- regulatory protein ca only bind to DNA in specific conformation
- regulatory proteins change their conformation after binding w/ effector molecule (Maltose)
- regulatory proteins requires maltose to bind at regulatory site -> is a positive regulator (binding = expression)
- e.g. of induction because genes required for maltose catabolism normally silent if no maltose, but induced in presence of maltose
Gene regulation; Prokaryotes
-lac operon - e.g. of negative regulator and induction
- regulatory protein binds at regulatory site w/out effector molecule
- is an example of negative regulator -> binding or regulator stops gene expression
- example of induction because genes required for lactose catabolism induced only in presence of lactose (remain silent in absence of lactose)
Telling if something is a positive or negative regulator and whether it is induction or repression
- Induction/repression: look and see what the effect of a small molecule (effector) is
- presence of molecule induces transcript = induction
- presence of molecule turns off transcript = repression - Positive/negative regulation: see what regulatory protein does
- if naturally binds and stops transcript = neg
- if naturally binds and helps transcript = positive
The lac Operon - lacI as a negative regulator
-In presence of lactose and without
a) Lac operon w/out lactose
- LacI repressor binds at operator site & maintains lac operon in inactive condition (Operator lies between promoter & structure genes)
- prevents RNApoly from initiation transcription
b) Lac operon in presence of lactose
- Inducer binds to repressor and converts it to inactive form
- inactive repressor can’t bind to operator -> RNA poly allowed to move forward for transcription
The lac Operon - cAMP-CAP complex acts as positive regulator
-How it works as a positive regulator
- Catabolite activator protein (CAP) binds to promoter of lac oepron and stimulates transcription
- must bind w/ adenosine-3’, 5’-cyclic monophsophate (cAMP) before binding
- binding of cAMP-CAP to promoter activates transcription by facilitating binding of RNA polymerase
- lvls of cAMP inversely related to glucose - low glucose stimulates high cAMP (vice versa)
- ensures that cell uses glucose first, even if lactose present
Gene regulation by DNA bending - e.g. of CAP protein
- Active CAP recognizes specific seq of DNA for binding
- CAP binding induces DNA bending
- introduces sharp kink w/in binding sequence (results in 90 degree bend)
- CAP functions by interacting w/ RNA poly (alpha subunit)
- introduces sharp kink w/in binding sequence (results in 90 degree bend)
is very important to start transcription
Gene regulation by DNA looping - using ara operon as an e.g.
- Structure of ara operon
- In Absence of arabinose
- in presence of arabinose
- ara operon has 3 structural genes: araB, araA, araD
- regulatory gene = araC
- 4 regulatory regions: araO1,araO2, araI1 & araI2
a) Absence of arabinose
- AraC binds to I1 & O2 - causes DNA looping
- results in no transcription from BAD and C
b) Presence of arabinose - Arabinose interacts w/ AraC - changes affinity
- AraC binds at I2 and of CAP at it’s binding site is required for transcription
*AraC acts as both positive & negative regulator
Gene regulation by attenuating transcription - using trp operon as e.g.
- Structure of trp operon
- When tryptophan levels are low
- trp operon has 5 structural genes: trpE, D, C, B, A
- control region has promoter, operator, leader peptide coding region (L) & attenuator
- region L locate between operator and attenuator
- control region has promoter, operator, leader peptide coding region (L) & attenuator
- tryptophan is needed by the cell!
a) Tryptophan lvls low - RNA poly beings to transcribe DNA, producing region 1 of 5’ UTR
- ribosome attaches to 5’ end of 5’ UTR and translates region 1 while region 2 being transcribed
- ribosome stalls at trp codons because lvls low, region 2 is not covered by ribosome when region 3 transcribed
- when region 3 transcribed, paired w/ region 2. When region 4 transcribed, it cannot pair w/ region 3 (as already paired w/ region 2); attenuator never forms and transcription continues
Gene regulation by attenuating transcription - using trp operon as e.g.
-When tryptophan levels are high
b) Tryptophan lvls high
- RNA poly beings to transcribe DNA, producing region 1 of 5’ UTR
- ribosome attaches to 5’ end of 5’ UTR and translates region 1 while region 2 being transcribed
- RNA poly transcribes region 3. DOES NOT stall at trp codons, because it is abundant
- ribosome covers part of region 2, preventing it from binding w/ region 3. Region 4 transcribed and pairs w/ region 3, producing attenuator that terminates transcription
Regulation of Eukaryotic Gene expression
-At genome level (2 ways)
- Can be controlled on many different levels
1. Regulation at Genome level
a. Chromatin decondensation: chromosomal puffs are regions of relaxed chromatin where active transcript. taking place
- chromatin usu v. compacted and protected (so that DNAse 1 doesn’t effect it)
b. Histone Modification: Histones modified by acetylation of lysine, phosphorylation of serine and threonine and methylation of lysine and arginie residues- acetylation causes decrease in overall positive charge = protein repels DNA (leads to expression of gene)
- acetylation of H3 & H4 associated w/ active chromatin
- methylation associated w/ inactive chromatin
Regulation of Eukaryotic Gene expression
- Transcriptional Level
- things that interact
- Enhancers and insulators
Transcriptional apparatus
*Initiation of transcription regulated by transcription factors and regulator proteins
-transcriptional activators & coactivators, transcriptional repressors (that bind to silencers), Enhancers & insulators
Enhancers: DNA seq. stimulating transcript. from a distance away from promoter
Insulator: DNA seq. that blocks or insulates effect of enhancers
Transcription apparatus: has RNA poly + transcriptional factors
Regulation of Eukaryotic Gene expression
-Transcriptional Activator proteins
- Transcriptional activator proteins bind to sites on DNA and stimulate transcription
- most act by stimulating or stabilizing assembly of basal transcription apparatus - different transcriptional activator protein binds to each consensus seq., so each promoter responds to unique combo of activator proteins
Regulation of Eukaryotic Gene expression
-Transcriptional level - GAL4 in response to galactose
-GAL4 binds to UAS(g) site and controls transcription of genes in galactose metabolism
-GAL4 must interact w/ basal transcription apparatus to stimulate transcription
In absence of galactose, GAL 80 blocks GAL 4 from activating transcription
-when galactose present, it binds to GAL 3 and brings about a change in conformation of GAL80
-GAL 4 can now interact w/ basal transcription apparatus and stimulate transcription.
Regulation of Eukaryotic Gene expression
- DNA response elements
- what they do
- co-ordinate expression of nonadjacent, but related genes
- similarly to operons in bacteria
- each gene transcribed as single transcriptional unit
- multiple genes can be regulated by having response element in each gene
- approx. 200 bp of promoter and comprise of the half sites
-All genes w/ same response element can be expressed together by producing regulatory protein that binds to this element
Regulation of Eukaryotic Gene expression
-Activation of gene expression - different ways activators can be activated
-Gene expression requires activator - can be controlled in many different ways
- i.e. inactive condition active condition
- no protein protein syn
- inactive protein protein phosphoryted
- inactive protein protein dephosphrylated
- bound to inhibitor inhibitor released
- bound to inactive partner change of partner
Regulation of Eukaryotic Gene expression
How do regulatory proteins interact w/ each other and w/ DNA?
-2 types of domains in these proteins
- Structural motifs that allow regulatory proteins to bind to DNA & activate transcription
- are 2 types of domains in these proteins
1. Transcription regulation domain (activation domain)- these domains have;
- high proportion of acidic a.a. or
- large no or glutamine or
- large no of proline
2. DNA binding sites
- these domains have;
Regulation of Eukaryotic Gene expression
-Motifs that allow regulatory proteins to bind to DNA (4)
- Helix-turn-helix motif
- Zinc finger motif
- Leucine Zipper motif
- Helix-loop-helix motif
Regulation of Eukaryotic Gene expression
Structural motifs - Helix-turn Helix
- Helix-turn Helix domain
* is a DNA binding domain
- present in product of homeotic genes
- 3 helixes - 1 lies in major groove of DNA (other 2 lie above DNA)
- An N-terminal arm makes contct w/ minor groove
*has helixes in them
Regulation of Eukaryotic Gene expression
Structural motifs - Zinc Finger Domain
- Zinc finger domain
* DNA binding domain
- formed by binding of Zn atom by group of conserved a.a. -> makes finger-like structure
- number of fingers varies among different proteins
- C-terminal part of each finger forms alpha helix that binds to DNA
- N-terminal part forms beta sheet.
Regulation of Eukaryotic Gene expression
Structural motifs - Leucine Zipper
- Leucine Zipper: a domain for protein-protein interactions
- Is a stretch of aa w/ a leucine at every 7th position
- active form is dimer held by leu-leu interactions- 2 proteins interact via leucines
-the DNA binding domain allows it to bind to DNA - dimer binding allows transcription
Regulation of Eukaryotic Gene expression
Structure motifs - Helix-loop helix
- Helix loop helix
* domain for protein-protein interaction
- hydrophobic interactions allows 2 proteins to interact w/ each other
- will contain a basic region to interact w/ DNA- 2 proteins w/ helix loop helix can interact to form dimer
* In dimer, if one of proteins does not have the basic region, it cannot bind to DNA
- 2 proteins w/ helix loop helix can interact to form dimer
Regulation of Eukaryotic Gene expression
-5 ways level of protein can be controlled via translation
- Regulation of protein factors involved in translation
- Regulation by translational repressors
- Regulation by mRNA half life
- Regulation by translational coupling
- Regulation by tRNA levels
Regulation of Eukaryotic Gene expression
- Regulation of protein factors involved in translation
e. g. regulation by heme in developing erythrocytes HCI
if not enough heme, globlin won’t be made (globlin = protein)
- cell regulates production of globlin via heme levels
- HCI (Heme Controlled Inhibitor) is a protein kinase
- if no heme, HCI becomes active as protein kinase
- controls activity of eIF2 (required for initiation of translation)
- if no heme, HCI becomes active as protein kinase
- Phosphorylated eIF2 is inactive and can not start translation
- No heme = no translation
Regulation of Eukaryotic Gene expression
- Regulation by translational repressors
- regulator protein may bind to site on mRNA that overlaps ribosome binding site at initation codon
- binding of regulator may therefore block binding of ribosome to mRNA - translation will not occur
*proteins that bind to sequences w/in initiation regions of mRNAs may function as translational repressors
Regulation of Eukaryotic Gene expression
- Regulation by translational repressors
- Translational control of ferritin (an iron storage protein)
- Iron response element (IRE) is present upstream to start codon in ferritin mRNA
- IRE binding protein binds to IRE and doesn’t allow ribosome binding to mRNA - ferritin not synthesized.
- Iron binds to IRE binding protein & changes its ability to bind to IRE
- if IRE binding protein not bound to IRE, ribosome can bind and start translation.
Regulation of Eukaryotic Gene expression
- Regulation by translational repressors
e. g. of regulation of ribosomal protein synthesis
- In ribosomes, ribosomal proteins bind to rRNA - levels of rRNA and protein controlled v. tightly
- Ribosomal proteins bind to rRNA and also can ind to their own mRNA
- when rRNA is available, the r-proteins associtate w/ it, translation of mRNA continues
- When no rRNA available, r-proteins accumulate - binds to mRNA and prevents translation
Regulation of Eukaryotic Gene expression
- Regulation by mRNA half life
- Transferrin e.g.
- Transferrin receptors required for iron uptake
- IRE present at 3’ end of transferring receptor to mRNA
- IRE binding protein when bound to IRE protects mRNA from nuclease degradation
- mRNA degradation starts from CAP -> deadnylation reduces polyA tail thus affecting the interaction w/ the cap (translation requires loop like structure that is formed by PolyA tail and protein at cap)
- changes 1/2 life of mRNA
Regulation of Eukaryotic Gene expression
- Regulation by translational coupling
- operon has several genes encoded together
- translation stops then straight away finds start codon of next gene
- cell combines stop and start codons
- only found in bacteria
e. g. start codon of trp D is coupled w/ stop codon of trpE
- ensures equal lvls of trpD and trp E gene products synthesised
Regulation of Eukaryotic Gene expression
- Regulation by tRNA levels
- tRNA brings a.a. in translation
- in some a.a. there are greater than 1 codon
- each organism has bias for a particular codon - make more tRNA for preferred codon
- Some tRNAs will be at very low level because their codons occur rarely
- therefore takes longer for tRNA to be brought - affects translation by slowing down process
