BIL 250 - Lecture 10

Click HERE for your print copy.

Gene Expression

To control the products manufactured by a cell at any given time in its development or life cycle, feedback mechanisms must operate which allow the cell to

repress

2. induce the manufacture of a product not constantly made.

This control can be exerted at the level of

2. translation (i.e., protein not made from the mRNA transcript)

3. protein/enzyme function (enzyme disabled; cannot attach to substrate)

Definitions:

catabolism: breaking down.
catabolic operons such as lac (which produces the enzymes that digest lactose) are induced to be transcribed when a substance to be catabolized enters the cell.
anabolism: building up
anabolic operons such as trp (which produces the enzymes that manufacture tryptophan) are repressed when the cell is saturated with the product of the operon.

In an inducible system the cell manufactures a product only when needed

In a repressible system the cell usually manufactures a product, but shuts down production when the product isn't needed.

Gene expression systems may operate under either positive or negative control, and each type is often linked to the inducible and repressible systems described above, like SO.

For classic examples of gene expression control at the transcriptional level, we return to our old pal, E. coli

The lac operon - an inducible system
The trp operon - a repressible system

The lac operon

Terms to know:

operon

promoter - a region of DNA to which RNA polymerase attaches in preparation for transcription.

operator - a short DNA sequence, usually located just behind the promoter, to which an enzyme can attach.

With the genes on the lac operon, E. coli can produce three enzymes in order to process the energy in the sugar lactose. These enzymes are:

Beta galactosidase

catabolize lactose into glucose and galactose OR
isomerize lactose into allolactose.

2. Beta galactoside permease - which facilitates transfer of lactose into the cell and concentrates it in the cell

3. Beta galactoside acetyl transferase - this acetylates other galactoside sugars that may be in the cell (besides lactose), preventing Beta galacosidase from breaking them down (which can produce toxic side products and be wasteful--need to concentrate on lactose!)

The genes comprising the lac operon, and which code for the three enzymes, are known as "Z" (B-galactosidase gene), "Y" (permease gene) and "a" (transferase gene). All are under the control of the same promoter.

Just upstream of this lac operon is an independent gene, known as "i". It codes for a specific repressor protein capable of binding to the operator of the lac operon.

When the repressor is bound to lac's operator, RNA polymerase can't attach to it, and mRNA is not transcribed from the genes. No lactose-catabolic enzymes are produced.

The repressor is an allosteric (shape-changing) protein, which has a binding site for a {{{mystery molecule}}}. When bound, the mystery molecule changes the binding properties of the repressor, causing it to release from the lac operator.

In the E. coli lac operon, the inducer (mystery molecule) is an isomer of lactose....allolactose.

When E. coli is in the presence of milk sugars (lactose), the following events take place.

some of the lactose spontaneously isomerizes into allolactose.
The allolactose binds to the allosteric binding site of the repressor, causing it to release from the operator/promoter of the lac operon.
RNA p'ase can now attach, and the three enzymes necessary for lactose catabolysis are transcribed/translated.

This system operates under positive feedback: as long as there is lactose in the cell, Beta galactosidase will be isomerizing enough of it to keep all repressor proteins in the cell bound and off the operator of the operon. Transcription continues as long as there's enough lactose.

When lactose concentration in the cell decreases, allolactose diffuses off the repressors (following the gradient); Beta-galactosidase can now split these and use them for energy, too.

Unbound, the repressor protein returns to the operator and clamps back on. Transcription of the three enzyme genes is thus stopped.

Once transcription and translation of the operon has ceased, some of the manufactured enzymes will still remain in the cell for a while, though most will be degraded/inactivated. The remaining Beta-galactosidases (which last for a relatively long time) may be instrumental in the initial

lactose-->allolactose

isomerization upon the E. coli's next lactose meal.

How was the operon studied? You guessed it. With Mutants.

lac operon constitutive mutants exist (Gene expression is "constitutive" when it results in constant, unregulated production of a product) in which the three enzymes are constantly transcribed. These exist primarily in the laboratory, where they have been instrumental in allowing investigators to elucidate the specifics of this system.

Other mutants exist in which each of the three enzymes are non-functional. These, too, were instrumental in helping investigators figure out the lac operon.

Other mutants (e.g., mutant operator, mutant promoter etc.) have been used as well.

Artificially produced merozygotes (remember, these are "partial diploids") have allowed investigators to map these genes via the type of analyses we did earlier in the semester.

The type of repressor control we have described above is an example negative control: the attached repressor protein turns the gene "off".

However, under certain circumstances, the lac operon can also operate under positive control.

Here's how.

. Glucose is a more desirable sugar than lactose (which must be processed to provide glucose, at additional cost to the cell). If plenty of glucose is present, the cell has mechanisms in place to ensure that it doesn't "waste its time" making the enzymes to break down lactose, even if lactose is present.
Under ordinary circumstnaces, the lac operon does not have a very high affinity for RNA p'ase, even when the repressor is removed.
There is another site, adjacent to the lac promoter, called the cap site. It has an affinity for a protein known as the catabolite activator protein (CAP).
Only when the CAP protein is bound to the cap site does RNA polymerase have a high affinitiy for the lac promoter. When the promoter is so bound, transcription of the operon is said to be promoted. This is how it happens:
- When glucose concentration in the cell decreases, cyclic AMP forms a complex with free-floating CAP in the cell.
- The CAP-cAMP complex binds to the cap site, just upstream of the lac promoter. This makes it very attractive to RNA polymerase
  
  ******
- When glucose concentration in the cell is high, cAMP concentration is low. When this is the case, there is too little available cAMP for the CAP-cAMP complex to be formed in any significant concentration.
- Therefore, the lac operon is transcribed at a high rate only when...
When cAMP-CAP complex is bound to the cap site, it acts as a "super-inducer" of the lac operon. This type of interaction is one form of POSITIVE REGULATION: attachment of the protein complex induces transcription. (As opposed to the binding of the repressor protein, which inhibits transcription.)

The trp operon

The tryptophan (trp) operon is a repressible system.

Under normal circumstances, the cell is in constant need of tryptophan, an important precursor to many cellular products. This means that under normal circumstances, the trp operator is unbound, and constantly available to RNA polymerase.

When there's an excess of trp in the cell, however, some of it will bind to and activate a free-floating regulator protein (R).

The R-trp complex has a high affinity for the trp operator. When it is attached, it blocks the attachment of RNA p'ase, stopping transcription. The operon is thus turned "off". The protein is a repressor.

Because the product of the operon (the amino acid tryptophan) is involved in repressing the very gene responsible for its manufacture, tryptophan is said to be a corepressor.

When the cell's trp concentration is very low, trp will diffuse off the regulator protein. The "naked" R repressor loses its affinity for the operator, and detaches.

RNA polymerase ase can once again attach to the operon, transcribing the five genes coding for the five tryptophan-manufacturing enzymes.

In this condition, the operon is said to be derepressed until trp concentration rises enough to form active R-TRP, which will again repress the operon.

When gene transcription is moderated by the attachment of a protein to the operator, the gene/operon system is said to be operating under operator control.

However, the trp operon also illustrates how an operon can function under attenuator control.

Attenuator control of the trp operon

Yanofsky (1981) first reported the attenuator mode in the trp operon. This gene expression control functions at the level of translation. Similar modes have since been found in several other amino acid anabolism pathways.

The mRNA leader transcript transcribed from the DNA attenuator region has four complementary "velcro" regions which can bind together in various ways to form stem loops.

The particular configuration of the attenuator transcript determines whether the trp operon is turned "on" or "off":

Part of the leader mRNA transcript of this attenuator region (bases 27 - 68, named the "leader peptide gene") encodes a small polypeptide, only 14 amino acids long. The protein includes two adjacent tryptophans. (This should give you a hint.)
The leader transcript is translated in one of two ways, and this is directly related to the amount of tryptophan available in the cell (and hence, the number of activated tryptophanyl tRNA's ready to add tryptophan to the protein).
When the ribosome translating the attenuator region hits the double trp region on the mRNA, one of two things can happen:
II. If there is insufficient tryptophan in the cell to provide readily available tryptophanyl-tRNA, the ribosome has to sit and wait for trp-tRNA. It "idles," covering region 1. This lag time allows stem loop 2/3--known as the "pre-emptor stem loop"--to form. It prevents formation of the non-translatable 3/4 loop.
Stem loop 2/3 is translatable. When it forms, translation of the operon continues, the enzymes are manufactured, and more tryptophan can be made from that transcript.
Remember that, in prokaryotes, multiple ribosomes attach to any given mRNA transcript and translate it as it's being made. Hence, the formation of either loop will determine the level of translation that takes place on any given trp mRNA transcript until it is degraded by the cell.

When amino acids are scarce in general (i.e., the cell is protein starved), both stem loops form, creating an untranslatable region on the mRNA transcript. (why?)

Repression & attenuation are redundant, but both systems are used by the cell under different circumstances.

More Prokaryotic Control of Gene Expression at the level of Translation

Recall: Control of gene expression may affect

the amount of mRNA produced (transcriptional control)
the efficiency with which protein is translated from the mRNA transcript (translational control)

In prokaryotes, control of gene expression at the translational level is uncommon. (Why?)

mRNA lifespan in a prokaryote is only about 2 minutes; exonucleases rapidly degrade them.

mRNA transcribed but not translated is a waste of energy. If it ever existed at all in an ancestral prokaryote, it might have been maladaptive.

However, translational control in prokaryotes can occur in some cases. When it does, it may be in the form of

unusually rapid degradation of a particular mRNA transcript
RNA/RNA hybridization: Double stranded RNA cannot be translated. (In one form of this control, only a short section of the 5' end is double-stranded, preventing ribosome attachment.)
differential ribosome binding efficiency: consensus sequences on certain genes have a lower affinity for the 16S subunit, and so always have a lower rate of translation than other genes.
codon preference: the most common tRNA's in the cell are most readily available. Transcripts with rarer codon/tRNA sequences will be translated more slowly. (Evolutionary note: This may have been a pre-existing condition-- perhaps in many transcripts--but was a liability if found for the more vital enzymes needed in large quantities.
Stringent Response: This occurs when the prokaryote is starved for amino acids, and translation must cease to avoid cell death. Here's how it happens:
- If a bacterium is starved for amino acids, there will be relatively plentiful tRNAs around that are not activated with an attached amino acid.
- Still, even an inactivated tRNA has some chemical affinity for the translation complex (mRNA + ribosome).
- If a "naked" tRNA happens onto the A site (which is more likely to happen when the bacterium is aa starved, and there are lots of "naked" tRNA's floating around), the tRNA "idles" at the A site.
- An special enzyme known as the stringent factor recognizes the idling tRNA, and produces an unusual nucleoside phosphate in the rRNA. This is guanosine tetraphosphate (3'ppGpp5')
- This is the "idling reaction", and the Guanosine tetraphosphate is known as the "magic spot."
- The precise mechanism is unknown, but the 3'ppGpp5' apparently sends the bacterium into a state of suspended animation.
- Translation ceases until the bacterium gets a good protein meal.

Control of Gene Expression in Eukaryotes

In eukaryotes, as in prokaryotes, control of gene expression can occur at the level of

transcription
translation
protein activity (post-translation)

Unlike prokaryotes, eukaryotes (especially multicellular ones) must undergo an intricate sequence of gene expression events not only during regular metabolism, but also during development of cell types, tissues, organs and organ systems and general morphogenesis.

It is probable that eukaryotic mechanisms are at least analogous to prokaryotic controls, though we may never know if they are truly homologous.

The Hogness Box

One promoter common to all eukaryotic proteome genes is known as the core promoter or basal promoter. This sequence of seven nucleotides--TATAAAA--is called the TATA Box or Hogness Box.

A transcription factor complex known as IID (TFIID) binds to the TATA box by means of a TATA-binding protein (TBP). Additional proteins in the complex interact with each other.

The entire complex acts as an activator.

gene enhancer sequences similar to the E. coli CAP site discussed previously may thus be involved in the Hogness Box control of eukaryotic gene expression.

Transcriptional Control
Transcription can be

turned completely on or off
attenuated

There are three classes of control elements in eukaryotes:

The RNA polymerase II binding region (a.k.a. the core promoter)
cis-acting binding sequences that bind to proteins with RNA polymerase affinity, which in turn help bind RNA polymerase to a promoter.
enhancers and silencers
A cis-acting DNA sequence is a genetic factor that acts upon several elements involved in gene transcription that are on the same DNA molecule.
A trans-acting DNA sequence is a similar factor that affects elements located on different chromosomes.
- In eukaryotes, enhancers and silencers are DNA sequences reminiscent of the CAP site in the E. coli lac operon:
  They are located upstream of the promoter region, and have an affinity for specific activator or repressor proteins that make the gene either more or less attractive to RNA polymerase.
  (Note: Recall that eukaryotes have THREE RNA polymerases, RNA p'ases I, II and III. RNA p'ase II is responsible for manufacture of all mRNA, so when we discuss proteome genes, the RNA polymerase involved is RNA polymerase II.)
- An enhancer DNA sequence (or positive regulatory element) turns a gene ON. The mRNA-binding protein for which it has an affinity is called a positive regulatory protein, or activator. When the activator is bound to the enhancer, RNA polymerase is more highly attracted to the gene.
- A silencer DNA sequence (or negative regulatory element) turns a gene OFF or reduces its rate of transcription. The mRNA-binding protein for which it has an affinity is called a negative regulatory protein, or repressor. When the repressor is bound to the silencer, RNA polymerase cannot attach and transcribe the gene.
Note that the genes encoding activators and repressors can be relatively far away (e.g., located on different chromosomes) from the enhancers and silencers they affect (trans-acting), or they may be located on the same chromosome (cis-acting).
It is believed that the action of enhancers and silencers involves their ability to bind downstream segments of DNA, causing them to loop upon themselves. This, in turn, can physically affect the rate of transcription.

Here's an OVERVIEW.

Transcriptional control also can be exerted by the activity ofDNA-binding domains, proteins which bind directly to the DNA to affect its transcribability.

Nucleosomes and Eukaryotic Gene Expression

Some data suggest that a gene not actively being transcribed may have histones associated with its TATA box.

The attached histones may block attachment of RNA polymerase to the TATA box, which makes transcription impossible.

Histone/nucleosome winding of the DNA molecule may change (between cell divisions) with the gene activity in a particular cell or tissue, or with the developmental stage of a particular cell.

How does the histone-wrapped gene become active? One hypothesis suggests that attachment of protein to the enhancer sequence (perhaps during the later stages of DNA replication) somehow disrupts the nucleosome/DNA winding. In this protein-enhanced configuration, DNA can be transcribed and translated.

DNA Configuration and Eukaryotic Gene Expression

DNA can exist in one of several isomers.

B DNA

This is the most common DNA conformation found in vivo

It is relatively narrow and more elongate than A or Z forms

phosphodiester backbone runs in a smooth curve

The base pairs are almost perpendicular to the axis of the double helix.

The wide major groove easily accessible to various polypeptides that affect level of transcription.

Small kinks along the length of a B-DNA strand may represent areas where a transcription factor is bound.

B-DNA form is believed to be the conformation of actively transcribed genes.

A DNA

RNA-DNA hybrids and double-stranded RNA are most often found in this form.

the helix is shorter and of greater diameter than in B-DNA

phosphodiester backbone runs in a smooth curve

base pair H-bonds are slanted relative to the axis of the double helix

the major groove has become deep and narrow, making it less accessible to proteins that might affect transcription.

genes in this conformation are generally believed to be not actively transcribed.

Z DNA

helix spirals to the left, instead of the right, as it does in B and A DNA

this isomer can form in vivo, but function is not fully understood.

helix is more narrow and elongate than either A-DNA or B-DNA

major "groove" is so shallow as to no longer form a groove.

minor groove very narrow

base pairs nearly perpendicular to axis of double helix

phosphodiester backbone forms a zig-zaggiung pattern because of unusual sugar configuration.

genes in this configuration are believed to be transcriptionally inactive.

Recall: Z-DNA is stable in vitro when its cytosines are methylated (the 5-carbon H is replaced by CH₃)

methylation protects DNA from the action of endonucleases.

Hypotheses:

When being transcribed, DNA exists as B-DNA; when not being transcribed, it exists as Z-DNA.
Also, methylation may promote nucleosome wrapping; transcription takes place in un-nucleosomed DNA in some cases, as described previously.

Genetic Response of Multicellular Organisms to Relatively Rapid Environmental Changes

How are certain cellular products produced so rapidly in response to sudden environmental changes?

Hormones may behave as regulators in multicellular eukaryotes in which short-term gene activation is necessary.

by definition, a hormone is a substance produced in one area, and used in a different area, to which it has been actively transported.
example: various steroid hormones (there are, of course, a number of different steroids) bind to specific receptors in the cytoplasm (SHR = "steroid hormone receptor). This receptor/steroid complex then binds directly to the DNA, regulating transcription.
another example: other hormones may bind to specific receptors in the plasma membrane. This binding activates adenyl cyclase (also embedded in the plasma membrane) to convert ATP-->cAMP. cAMP affects the binding of the hormone to the DNA, which can affect transcription rate.

Post-Transcriptional Control

An mRNA consists of a 5' UTR (untranslated region, or leader), an ORF (open reading frame, a.k.a. polypeptide coding sequence) and a 3' UTR (untranslated region, or trailer).

mRNA Lifespan

Certain sequences in the 3' UTR may promote very rapid degradation.

Other UTR sequences may not promote degradation, but can lead to lower levels of translation.

Mutations in these sequences can result in higher-than-normal translation of proteins encoded in the ORF portion of the gene, which is how they were discovered.

Hence, regulatory information is encoded not only in the protein-coding regions, but in the non-protein coding regions of mRNA

mRNA Modification

There is some evidence to suggest that modification or non-modification of the primary mRNA transcript may be used as a form of post-transcriptional control.

The spliceosome (remember the snurps) may serve as a form of post-transcriptional modification, altering gene expression via alternative splicing.

When associated with the snRNPs, mRNA cannot leave the nucleus (it can't bind and pass through the nuclear pores).

When intron-bound snurps release the mRNA transcript, the spliced mRNA can exit the nucleus, but the introns can't.

If the number of transcripts actually allowed to move out of the nucleus and into the cytoplasm is regulated, transport control is said to be in effect.

Post-Translational Control

Once a protein is made, how is its activity controlled?

activity can be controlled if the protein is allosteric.
(since proteins generally have a longer lifespan than mRNA in the cell, it is to the cell's advantage to have some sort of protein manaagement mechanism in place)
often, the end product of an operon binds to the allosteric site of the operon's enzyme, changing its affinities and activity. (e.g., tryptophan)
when the concentration of the end product decreases, it diffuses off the bound allosteric proteins, allowing that protein to once again become active.

Protein Lifespan
Proteins can last in the cell from a few seconds to longer than a cell cycle. How does the cell know which ones not to break down?

The N-end Rule: The specific aa at the N-(amino)-terminal signals the polypeptide's lifespan to various proteases in the cell.
(e.g.- arg lasts about 2 minutes; met lasts up to 20 hours)
The PEST Hypothesis: The # of regions rich in the four amino acids proline (P), glutamate (E), serine (S) and threonine (T) signal lifespan to the various proteases. Proteins rich in these regions have lifespans of 2 minuts or less; in almost all proteins with lifespans over 20 hours long (some as long as 220), the PEST sites are lacking.

Epigenesis

In Genetics, Epigenesis is a heritable modification in gene function that occurs without change in the actual base sequence of the DNA code.

Paramutation: Anthocyanin pigment in corn plants

paramutation - the genetic activity of a particular allele is reduced when coupled with inheritance of a paramutagenic allele at the same locus.

Anthocyanin pigment is coded by an allele called "B"
A recessive mutant (b) results in a null allele: no pigment is produced
A paramutagenic allele at this locus (B') causes reduced pigment production.
BB x B'B' F1 heterozygotes are able to make only the reduced amount of pigment encoded by the B' allele. The BB' hybrid is indistinguishable from the B'B' homozygote.
So does that mean B' is dominant to B?
It's more complex than that.
If you cross the F1 plants, all offspring exhibit the weakly pigmented phenotype--and all subsequent generations exhibit only the B' phenotype.
The B allele is apparently permanently "crippled" in its activity by being inherited along with the B' allele. It has paramutated.
The exact mechanism is not fully understood.

Parental Imprinting

parental imprinting: a gene inherited from one or the other parent is permanently inactivated, and is inherited and passed on in the same condition.

Human disorder examples

Note:

Paternally imprinted autosomal genes are expressed as if they are hemizygous, even though there are two copies of each in every cell.

DNA sequencing reveals no change in the DNA sequence of imprinted genes.

Rather, some of the bases of the inactivated gene are methylated. As we already know, there is a correlation between methylation and rate of transcription: highly methlylated genes are transcribed at a far lower rate than genes with low levels of methylation.

(Caution: correlation does not imply cause and effect. It is not yet known whether the methylation of DNA causes epigenetic effects, or if the altered methylation is caused by the epigenetic effects.)

Re-thinking a Classic Tale of Natural Selection: Epigenesis in Pepper Moths?
Recall the tale of Biston betularia, the Pepper Moth, still a "poster child" for teaching the mechanism of natural selection.

...but is the classic tale really what happened in Nature?

Some researchers suspect that industrial melanism is actually induced by environmental factors and becomes heritable, making it a case of epigenesis. The exact mechanism of the pigment genes activity being altered in a heritable way is not yet known.

Epigenesis may simply be cases of gene expression gone (somewhat) awry. In most cases, the exact mechanism of epigenetic change that produces phenotypic change is not yet well understood.

Could epigenesis be a source of exaptation? An epigenetic character might allow the eukaryote exhibiting it to quickly alter the genome (using pre-existing material, and merely "repackaging" it) so that offspring are phenotypically different and (in some cases) better suited to a changed environment than the parental generation.

Stay tuned.