15 regulation of gene expression expression of genetic information is dependent on regulatory...
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15 Regulation of Gene Expression
Expression of genetic information is dependent on regulatory mechanisms that either activate or repress the transcription of genes. Transcription is modulated by theinteraction at various regulatory molecules with DNA sequences, most often located upstream from affected genes.Genetic regulation in eukaryotes also occurs during post transcriptional events.
Gene expression• Transcription
– Initiation Elongation Termination
Francois Jacob
•Translation–Initiation
–Elongation
–Termination
Jacques Monod
• 15.1 Genetic Regulation in Prokaryotes:An Overview15.2 Lactose Metabolism in E. coli: An Inducible SystemStructural GenesThe Discovery of Regulatory MutationsThe Operon Model: Negative'ControlGenetic Proof of the Qperon ModelIsolation of the lac RepresserThe CAP Protein: Positive Control of the lac Operon
• 15.3 Tryptophan Metabolism in E. coli:A Repressible Gene SystemGenetic Evidence for the trp Operon ,Attenuation15.4 Genetic Regulation in Eukaryotes:An Overview
• 15.5 Regulatory Elements, Transcription Factors, and Eukaryotic GenesPromotersEnhancersTranscription FactorsStructural Motifs of Transcription FactorsAssembly of the Transcription ComplexChromatin Conformation, DNA Methylation,and Gene Expression15.6 Gene Regulation by Steroid Hormones15.7 Posttranscriptional Regulation of GeneExpression:Alternative Splicing of mRNA
15.1 Genetic Regulation in Prokaryotes:An Overview
• Regulation of gene expression has been studied extensively in prokaryotes, particularly in Escherichia coll. Highly efficient mechanisms have evolved that turn genes on and off, depending on the cell's metabolic needs in particular environments. Detailed analysis of proteins in E. coll has shown that for the more than 4000 polypeptide chains encoded by the genome, a vast range of concentration of gene products exists. Some proteins may be present in as few as 5-10 molecules per cell, whereas others, such as ribosomal proteins and the many proteins involved in the glycolytic pathway, are present in as many as 100,000 copies per cell. The idea that microorganisms regulate the synthesis of gene products can be illustrated by considering the utilization of lactose (a galactose-glucose-containing disaccha-ride) as a carbon source. When it is present in the growth medium, many bacteria and yeast produce enzymes specific to lactose metabolism. When lactose is absent, the enzymes are not manufactured. These organisms thus "adapt" to their environment, producing certain enzymes only when specific chemical substrates are present. Such enzymes are said to be inducible, reflecting the role of the substrate, which serves as the inducer of enzyme production. In contrast, other enzymes that are produced continuously, re-gardless of the chemical makeup of the environment, are described as constitutive.Studies have also revealed cases where the presence of a specific molecule causes inhibition of genetic expression. This is often the case for molecules that are the end products of biosynthetic pathways. Amino acids can be synthesized by bacterial cells, but if the amino acids are present in the growth medium, they can be taken up and used. In such cases, it is inefficient for the cell to produce the enzymes necessary for the synthesis of those amino acids, and transcription of mRNA for the appropriate biosynthetic enzymes is repressed. This is an example of a repressible system of gene regulation.Regulation, whether it is inducible or repressible, may be under either negative or positive control. Under negative control, genetic expression occurs unless it is shut off by some form of a regulator molecule. In contrast, under positive control, transcription occurs only if a regulator molecule directly stimulates RNA production. In theory, either type of control can govern inducible or repressible systems. Our discussion in the ensuing sections of this chapter will clarify these contrasting systems of regulation. For the enzymes involved in lactose and tryptophan, negative control is operative.
Regulation of Gene Expression
• At Transcription stage:– Economical, but can’t be reversed quickly
• At Translation stage:– Wasteful, but easily reversible
Regulation of Gene Expression
Feedback regulation:
– Positive control: the gene is off unless it is turned on
– Negative control: the gene is on unless it is turned off
Regulation of Gene Expression
Feedback regulation:
– Induction: the “target” molecule turns expression ON (e.g., by disactivating the repressor)
– Repression: the “target” molecule turns expression OFF
The lac Operon in E. coli
Genetic System under both Induction and Repression regulation.
Let’s first consider Induction.
Cis- and trans- acting regulatory sequences
• Operator: a cis-acting elementInfluences expression of genes downstream
from it on the same 2-stranded DNA
• Repressor gene: a trans- acting regulatory geneInfluences expression of any relevant genes
in the same cell, on the same or different DNA
Let’s stop here and solve some problems
• You have 2 constitutive lac mutants (always expressing lac operon). You transform them with a plasmid containing an intact lac operon. One of them changes the phenotype to wild type. The other does not no matter how much you try. What may be the genotypes of these 2 mutants?
Let’s stop here and solve some problems
• You have a mutant strain with a mutation in lac operon repressor gene, which prevents lactose from binding to repressor protein.
• What will be the phenotype? Can it be changed by transforming this strain with a plasmid containing any lac operon structural or regulatory genes?
Homework!
• Textbook, Ch. 17, p.459: #5, 6, 10(extra spicy, optional)
• Problem manual, p.82-83: #44, 48 (solved)
Pop-up quiz:
Genotype Phenotype
(lactose present)
Phenotype
(lactose absent)
I- P+ O+ Z+(I- codes for inactive repressor protein)
I- P+ O+ Z+/
I+ P+ O+ Z-
CAP-cAMP control
Glucose present ->
cAMP level decreases ->
no CAP-cAMP complex is formed ->
no CAP binding to promoter ->
no activation of transcription
Let’s compare two controls of lac operon
• Repressor-operator control
• CAP-cAMP control
Let’s compare two controls of lac operon
• Repressor-operator control
Negative control
Control molecule: lactose
• CAP-cAMP control
Positive control
Control molecule: glucose
Another operon under double control:
Trp operon
Trp = triptophan
Let’s compare regulation of of lac operon and trp operons
• lac operon
Induction (target molecule, lactose, induces expression by desactivating repressor protein)
• trp operon
Repression (target molecule, triptophan, represses expression by activating repressor protein)
But.. It’s not the whole story!
Attenuation regulation of trp-operon
Regulation of Gene Expression in Prokaryotes
Hartwell pp. 551-560, 567-571
Regulation of transcription
• Differences in the basepairs in the -35 and -10 boxes allow genes to be expressed at different levels.
• This kind of regulation is important, but it does not allow the cell to adjust its pattern of gene expression in a dynamic manner to meet changing needs and environmental stresses.
Strategies for regulating gene expression in prokaryotes
• Switches in the subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
Strategies for regulating gene expression in prokaryotes
• Switches in the subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
Switches in the subunit of RNA polymerase
• Under certain conditions, the cell needs to induce a large set of genes that are normally silent
• Examples include:– heat-shock proteins– nitrogen starvation– developmental changes such as sporulation
Switches in the subunit of RNA polymerase
• Under certain conditions, the cell needs to induce a large set of genes that are normally silent
• This can be accomplished by synthesizing or activating a different RNA polymerase subunit that recognizes a distinct set of promoter sequences.
70 -32 -
54-
pg. 786
Strategies for regulating expression in prokaryotes
• Switches in the subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
In prokaryotes, genes with related functions can be expressed as a single mRNA controlled by one promoter
O LacY LacA
lactose permease-galactosidase unknown function
P
Promoter
LacZ
Transcription
The lac operon
Operons are regulated by two kinds of elements
Operator
Transcription
Promoter Gene 1
RNAP Transcriptional regulatory proteins
Regulatory sites
Transcriptional regulatory proteins
• Often called transcription factors• Some transcriptional regulatory proteins
stimulate transcription initiation (called activators)
• Other transcriptional regulatory factors inhibit transcription (called repressors)
• In bacteria, the sites where transcription factors bind reside close to the transcription start site. These binding sequences are called operators.
The regulatory gene for an operon can reside at another site of the chromo
some
pg. 869
The Lac Operon:A classic example of dynamic regulation of g
ene expression
• -galactosidase
pg. 868
The addition of lactose to E. coli leads to a large increase in -galactosidase synthesis
-gal synthesis is approximately6% of total protein synthesis
-gal synthesis stops
(cell growth)
- -galactosidase expression is inducible.
pg. 869
The addition of lactose to E. coli leads to a large increase in -galactosidase synthesis
-gal synthesis is approximately6% of total protein synthesis
-gal synthesis stops
(cell growth)
-Note: The increase in -galactosidase synthesis only occurs in the absence of preferred carbon/energy sources such as glucose.
pg. 869
Isolation of mutations affecting the induction of the lac operon
Inducers of the lac operon
1,6-Allolactose
IPTG
-the natural inducer of lac operon expression-formed from lactose by an activity of -galactosidase
-a synthetic inducer of lac operon expression-cannot be metabolized
Isolation of mutations affecting the induction of the lac operon
Assaying for -galactosidase activity using X-Gal substrate
Isolation of mutations affecting the induction of the lac operon
Assaying for -galactosidase activity using X-Gal substrate
wild-type E. coli grown on mediawith X-gal but without glucose
wild-type E. coli grown on mediawith X-gal and IPTG but without glucose
Isolation of mutations affecting the induction of the lac operon
mutagenize
plate on mediawith X-gal and
lacking IPTG andglucose
a mutant that expresses-galactosidase without inducer
Constitutive mutant
Isolation of mutations affecting the induction of the lac operon
mutagenize
plate on mediawith IPTG, X-gal
and lacking glucose
a mutant that fails to expressfunctional -galactosidase
• Most of these mutation have inactivated the lacZ gene rather than affected its expression.
• However, there are mutants in which the expression of lacY and lacA have also been lost.
Uninducible mutant
Production of B-galactosidase by strains grown under different conditions
IPTG - - + +
Glucose - + - +
Strain:
WildType no no yes no
LacZ- no no no no
oc yes no yes no
i- yes no yes no
is no no no no
Genetic analysis of lac operon regulation identifies two key components
• Transcriptional regulatory protein– encoded by the lacI gene (maps near the lac operon)
– loss-of-function lacI (i-) mutations cause the lac operon to be constitutively expressed
• This indicates that LacI acts as a repressor of lac operon transcription in the absence of inducer
• Site for lac repressor binding– called the lac operator (lacO)
– mutations of the lac operator (oc) also lead to failure of lac repressor binding and constitutive expression
Regulation of the lac operon
pg. 872
Using bacterial merodiploids to perform complementation tests
• E. coli are haploid,
• but some E. coli have large plasmids called F factors that can be transmitted (mated) from one E. coli to another.
• Through complicated genetic tricks, it is possible to isolate strains in which the F factor contains a large region of E. coli chromosome including the lac operon.
• Such a partially diploid strain is called merodiploid.
F factor
lac lac
E. coli chromosome
Transcriptional regulatory protein
Operator
Transcription
Promoter Allele 2
Operator
Transcription
Promoter Allele 1
Cis vs. Trans
• Trans-acting: In a cell with two copies of an operon, the transcriptional regulatory protein will regulate the expression of either allele,
• Cis-acting: An operator (the site where a transcriptional regulatory protein binds) can only affect the allele to which it is joined.
F factor
lacI-, lacO+, lacZ+ lacI+, lacO+, lacZ-
Evidence that lacI encodes a repressor: the LacI protein can act in trans
• Result: Inducible synthesis
Constitutive Uninducible
Evidence that lacO acts in cis
F factor
lacI+, lacO-, lacZ- lacI+, lacO+, lacZ+
F factor
lacI+, lacOc, lacZ+ lacI+, lacO+, lacZ-
inducible
constitutive
• Dominant mutations that gave uninducible lac operon expression were also isolated in the lacI gene.• These encode a “super repressor” (is) that cannot bind the inducer and therefore is always bound to t
he operator
Biochemical analysis of LacI repressor action
• Identification of the lac operator sequence
Biochemical confirmation that LacI repressor acts by binding to the lac operator in the absence of the inducer
QuickTime™ and aPhoto - JPEG decompressor
are needed to see this picture.
Hartwell, pg. 540
Lac repressor binds the operator as a dimer
The lac repressor binding site overlaps the RNA polymerase and tran
scription initiation sites
Hartwell, pg. 543
Summary of analysis of LacI repressor action
• Results– Lac repressor binds the operator region.
– Constitutive mutants • change the operator so that it cannot bind repressor (oc)
• change the repressor so that it cannot bind the operator (i-)
– The addition of inducer prevents lac repressor from binding the operator.
– Uninducible mutants (is) change the repressor so that it cannot bind inducer (and therefore stays bound to the operator)
Strategies for regulating expression in prokaryotes
• Switches in the subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
Catabolite Activator Protein
• Even in the presence of lactose, the lac operon is poorly expressed if a preferred carbon/energy source such as glucose is present.
• The lac operon promoter is very weak and requires another transcriptional regulatory protein to be strongly expressed.
• This protein is called catabolite activator protein (CAP).
O LacY LacAP LacZ
Transcription
LacI
Catabolite activator protein• E. coli has a system for monitoring its energy levels.• When energy stores are low, the enzyme adenylcycl
ase catalyzes the synthesis of cyclic AMP from ATP.
• cAMP serves as a signal to turn on operons responsible for digesting (catabolizing) less-preferred sugars such as lactose.
Catabolite activator protein
cAMP
CAP
CAP
cAMP
Transcriptional activator
Catabolite activator protein
• CAP-cAMP binds to the promoter next to the RNA polymerase binding site.
• CAP makes contacts with RNA polymerase that increase the binding of RNA polymerase to the lac promoter.
Hartwell, pg. 543
lactose and glucose
no lactose or glucose
lactose without glucose
glucose without lactose
Off
Off
Off
On
Strategies for regulating gene expression in prokaryotes
• Switches in the subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
The trp operon: a classic model for attenuation
• The trp operon encodes the enzymes required to synthesize tryptophan (TrpA-E).
• Synthesis of the trp mRNA is controlled by a repressor that blocks transcription when bound to tryptophan.
• Therefore, when tryptophan levels are high, transcription will be repressed.
The trp operon
Hartwell, p. 546
The Trp operon is regulated by a repressor
Hartwell, p. 546
• However, in the absence of trp repressor, the synthesis of trp mRNA is still partially repressed by the presence of tryptophan in the growth medium.
Hartwell, p. 546
Attentuation: The trp operon mRNA contains a leader region that can fold into different stem-loop structures
Trp codons in the leader sequence sense the availability of tryptophan
• The trp mRNA leader contains two trp codons in a row.
• When the tryptophan supply in the cell is limited, ribosomes will stall at these trp codons.
• The ability of the ribosome to read through these codons regulates the stem-loop choice.
The availability of tryptophan regulates which stem loop structure forms
Other operons encoding amino acid biosynthesis enzymes are also regulate
d by attentuation
p. 888
Strategies for regulating gene expression in prokaryotes
• Switches in the subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
Regulation of Gene Expression in Eukaryotes
Hartwell pp. 250-256, 581-590, 602-609
Transcription and translation in prokaryotes vs eukaryotes
• In prokaryotes, transcription and translation are tightly coupled.
• In contrast, transcription and translation are spatially separated in eukaryotes.
Primary RNA transcripts are extensively modified before leaving the nucleus
• Nascent mRNA is “capped” with a 7-methylguanosine at the 5’ end
• 3’ end is polyadenylated (100-200 residues) to protect against degradation
• mRNA editing can occur in rare cases• Splicing: often introns must be removed from tra
nscript and protein-coding exons spliced together to form mature mRNA for translation
Primary RNA transcripts are extensively modified before leaving the nucleus
• 5’ capping– protects mRNA from ribonucleases– promotes translation– Capping enzyme adds backward G– Then methyl transferases modify G
and 1 or more nucleotidesFirst base of transcript
Primary RNA transcripts are extensively modified before leaving the nucleus
• 3’ polyadenylation– Cleavage and polyA additio
n are initiated via recognition of AAUAAA site 11-30 nucleotides upstream of tail
– Protects RNA from rapid degradation
– May aid transport out of nucleus
– May increase efficiency of translation
Primary RNA transcripts are extensively modified before leaving the nucleus• mRNA editing
– only a few examples are known
synthesized in the intestine
deaminase is onlypresent in small intestine
synthesized in the liver
Primary RNA transcripts are extensively modified before leaving the nucleus
• mRNA splicing• Some parts of the RNA t
ranscript are removed prior to translation
• the excised pieces are called introns (intervening)
• the remaining pieces are called exons (expressed)
The mechanism of RNA splicing
• Certain sequences are conserved near splicing junctions and serve as splicing signals
• Some key bases are in the intron and others are in the flanking exons
The mechanism of splicing
• Splicing involves the excision of the intron in a lariat form.
The mechanism of splicing
• Splicesomes are large RNA- and protein-containing complexes.
• Some of the RNAs are used to recognize the splice junctions.
• Example: U1 snRNA recognizes 5’ splice site
The mechanism of splicing
• Splicesomes are large RNA- and protein-containing complexes
• Some of the RNAs are used to recognize the splice junctions
• Example: U2 snRNA recognizes branch site
Spliceosome Assembly
Why splicing?
• mRNA splicing can allow a single gene to express several different proteins (isoforms)
• These different forms often have different activities, can interact specifically with different molecules– For example, splicing can result in secreted vs membrane
bound forms of a protein like an antibody
• The production of different isoforms can be regulated during development– The Drosophila sex determination system provides several
classic examples of developmentally regulated alternative splicing giving rise to differing functions
Splicing can produce a secreted vs membrane-bound antibody
• Alternative splicing of this heavy chain gene can produce two different types of antibody with the same specificity
• When activated by antigen, B-cells begin secreting their antibodies
• Secreted form lacks exons 7 and 8 which encode membrane attachment domain
2 63 541
Translation startStop Stop
Doublesex female isoform
Doublesex male isoform
activates female-specific genesand represses male-specific genes
activates male-specific genesand represses female-specific genes
Drosophila Doublesex is alternatively spliced into male or female forms
Functional Transformer
No functional Transformer
Regulation of transcriptional initiation in eukaryotes
• Three different RNA polymerases transcribe genes in eukaryotes– RNA polymerase I transcribes rRNAs (18S, 5.8
S and 28S)– RNA polymerase II transcribes mRNAs and sn
RNAs– RNA polymerase III transcribes tRNAs and 5s r
RNAs
• Each polymerase consists of many subunits.
Promoter elements in prokaryotes
• In prokaryotes, the promoter elements are well-defined.
• They are always present and always in the same position relative to the start site of transcription.
Promoter elements in eukaryotes
• In eukaryotes, the promoter elements are much less well-defined.
• They are not always present and can be various distances from the start site of transcription.
usually present
frequently not present
Transcription factors TFIIA, B, D, E and F play important roles in transcript in
itiation by RNA polymerase II
• These factors were identified by purifying the proteins required for correct initiation in vitro.
• Each factor is composed of multiple proteins.• TFIID contains the 30 kilodalton (kD) TATA-box
binding protein.• This complex of proteins together is called the bas
al transcriptional machinery.
Sequentialassemblyof thebasaltranscriptionmachinery
Enhancer elements control the activity of eukaryotic promoters
• Eukaryotic promoters are invariably weak and require “enhancer elements” to drive high level expression.
• Enhancer elements are defined as DNA sequences that promote the expression of a gene.– Enhancer elements are distinguished from promoters in that t
heir function does not depend on their orientation relative to the transcription units.
(TATA)
Enhancer
(TATA)
Enhancer
or
Enhancer elements control the activity of eukaryotic promoters
• Enhancer elements do not need to be placed at a precise distance from the promoter.
• Enhancer elements are frequently tens of kilobasepairs (Kb) away from the transcriptional start site.
(TATA)
Enhancer
or
(TATA)
Enhancer
Enhancer elements contain multiple binding sites for transcriptional activator proteins
• Many developmentally important genes have enhancers with binding sites for many different transcriptional activator proteins.
• Transcriptional activator proteins frequently consist of two modular domains:
• Many transcriptional activators have negatively-charged regions that may interact with components of the basal transcription machinery.
• Removing the activation domain can convert a transcriptional activator into a repressor
DNA looping allows transcriptional activator proteins to interact with the TFII components
• Silencer elements that provide binding sites for transcriptional repressor proteins also exist
Repressor proteins reduce transcription through competition or quenching
• Repressor can compete with activator for enhancer site
• Repressor can quench activator by– Blocking DNA-binding
domain– Blocking activation
domain
The expression pattern of an individual gene is determined by the transcription factors th
at bind its enhancers and their regulation
How is the activity of enhancers regulated during development and in respo
nse to environmental changes?• Many types of regulation are observed:
– tissue-specific expression• Many transcriptional activators are only expressed in specific cell types.
– by binding to hormones• For example, steroid receptors are enhancer binding proteins whose activity d
epends on hormone binding.
– by phosphorylation• Many transcription factors are only active when phosphorylated. These inclu
de transcription factors that respond to hormones and growth factors such as insulin.
• Together, these kinds of regulation can limit gene expression to specific times and tissues.
The GAL4, GAL80 Gene System
• One enhancer controls the genes GAL1,7, and 10
• Its activator, GAL4, is quenched by GAL80 in absence of galactose
• GAL1 and GAL3 can be induced by galactose to bind GAL80 and prevent it from blocking GAL4
• This allows GAL1,7, and 10 to be transcribed at high levels
• Heat Shock(HS) driven Flp enzyme causes recombination between homologous FRT sites during mitosis
• This derepresses a tissue-specific Gal4 transgene by recombining away Gal80 repressor
• Leading to clones of cells positively marked by the membrane marker CD8-GFP
Lee and Luo, Neuron 1999
Mosaic Analysis with a Repressible Cell MarkerMARCM
nc82CD8-GFP
MARCM can label a single neuron in the fly brain
Two types of regulation that we will not have time to discuss
• Regulatory proteins that act by modifying chromatin structure to allow transcription– Nucleosomes block the promoters of most inactive genes.
– Some proteins such as the SWI-SWF complex in yeast act by modifying chromatin structure to give the basal transcription machinery greater access to promoters.
• Regulation of mRNA accumulation by controlling mRNA stability– Length of poly-A tail
– RNA-binding proteins that protect RNA from degradation
Regulation of Gene Expression in Eukaryotes
Chapter 21
Eukaryotic gene regulation is different from regulation of prokaryotic genes
• Complex chromosome structure• Multiple chromosomes• Spatial & temporal separation of
transcription and translation• Transcripts processed in nucleus• mRNA lasts longer• Must have translational controls• Multicellular with many cell typ
es
Eukaryotic genes have promoters and enhancers
promoterenhancer
Eukaryotic genes vary in the number and arrangement of controlling elements
Mutations in the Promoter Region Can Drastically Alter Transcription
Overview of initiation of eukaryotic transcription
•TBP of TFIID binds ~20bp•IIA & IIB Bind•RNA Polymerase II, IIF binds•IIE,IIH, IIJ bind•Promoter clearance occurs•basal transcription ensues
Enhancers control chromatin structure and transcription rate
•Different from promoters because….•Position need not be fixed•Orientation is not critical•Introduced enhancers speed up transcription
How do they work?
Enhancers cause loops in the DNA which allow TFs to interact with transcription complex
Stabilizes transcription complex
Helix-turn-helix
Transcription factors have functional domains that permit binding to DNA and others that facilitate binding to protein in transcription complex
True activator transcription factors include….
Zinc Fingers
and…..
Leucine Zippers
and finally…...
Antirepressor Transcription factors
• Change chromatin structure to allow polymerase binding
•Several processes involved
ATP dependent remodeling (SWI/SNF)
Histone modification by Histone Acetyltransferase Enzymes (HAT)
•Acetylation of basic amino acids of histone lessen interaction with DNA•Deacetylases (HD) reverse this
Methylation of CG doublets regulates gene expression•Expression high when methylation low•tissue specific
Alternative splicing of mRNA occurs in different tissues•E.g. preprotachykin gene product
Controlling mRNA stability
• General or cell specific
• Stability sequences
• Instability sequences (e.g. AUUUA on fos mRNA)
• Address sequences
• Translational controls
Translation controls can be used to regulate final gene products
•Tubulin gene regulated by tubulin subunit conc. in cytoplasm (autoregulation)