Where to put the gene after eukaryotic promoter for best expression levels?

Where to put the gene after eukaryotic promoter for best expression levels?

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As far as I know there is an optimum distance between a promoter and the gene for the best expression levels. What is that distance for common promoters like CMV, SV40? If you have a first hand experience about this (e.g. if you have cloned the gene way downstream of the promoter and didn't/did see expression) and share it I will be grateful.

If you have comprehensive knowledge for other promoters please feel free to add them in the list too; they don't need to be common. I just want to hear people's experience on the subject.

I used pEYFP-N1 (stock vector) where the spacing between the CMV promoter and the EYFP start codon is ~88 nt, and the expression is high.

I also cloned a protein 5' of the EYFP, only 4 nt after the CMV promoter. The expression of this fusion was also high. Because I was only using the chimeric protein in experiments, I did not try to assess differences in EYFP expression.

If you look here: you'll see the basic pEYFP-N1 vector and the separation between the CMV promoter and the EYFP coding sequence.

On wiki: "The TATA element and BRE typically are located close to the transcriptional start site (typically within 30 to 40 base pairs)."

Tetracycline (Tet) Inducible Expression

To advance the study of gene function, scientists were in search of inducible promoters capable of controlling eukaryotic gene expression. Several endogenous promoters had been identified that responded to stimuli, such as hormones or metal ions however, these systems were confounded by secondary effects. Scientists began to pursue a non-endogenous system for eukaryotes. At the time, bacterial systems from E. coli presented the best candidates for inducible expression.

Bacterial systems were tested for functionality in mammalian cells. The lac system with the IPTG inducer was tested first, and IPTG was found to be inefficient, resulting in low levels of induction. In 1992, Manfred Gossen and Hermann Bujard tested the tet system in a mammalian cell system (HeLa) and found that the tet system was functional, and had rapid induction with efficient tetracycline uptake.

Tet System and the Tet Response Element (TRE)

A TRE is 7 repeats of a 19 nucleotide tetracycline operator (tetO) sequence, and is recognized by the tetracycline repressor (tetR). In the endogenous bacterial system, if tetracycline, or one of its analogs like doxycycline, are present, tetR will bind to tetracycline and not to the TRE, permitting transcription.

Tetracycline-dependent promoters are developed by placing a TRE upstream of a minimal promoter.

B. Complexities of Eukaryotic Gene Regulation

Gene regulation in eukaryotes is more complex than in prokaryotes. This is in part because their genomes are larger and because they encode more genes. For example, the E. coli genome houses about 5,000 genes, compared to around 25,000 genes in humans. Furthermore, eukaryotes can produce even more than 25,000 proteins by alternative splicing of mRNAs and in at least a few cases, by initiating transcription from alternative start sites in the same gene. And of course, the activity of many more genes must be coordinated without the benefit of multigene operons! Finally, eukaryotic gene regulation is made more complicated because all nuclear DNA is wrapped in protein in the form of chromatin.

All organisms control gene activity with transcription factors that bind to specific DNA sequences (cis regulatory elements). In eukaryotes, these elements can be proximal to (near) the promoter of a gene, or distal to (quite far from) the gene they regulate. A eukaryotic map showing the components of a typical gene and its associated cis-acting regulatory elements is shown below.

Enhancers are typical distal cis elements that recognize and bind transcription factors to increase the rate of transcription of a gene. Oddly enough, these short DNA elements can be in the 5&rsquo or 3&rsquo non-translated region of the gene, or even within introns, and can lie thousands of base pairs away from the genes they control. Note that enhancer elements are even in introns can also be very far from the start-site of transcription of a gene.

Upstream regulatory regions of eukaryotic genes (to the left of a gene promoter as shown above) often have distal binding sites for more than a few transcription factors, some with positive (enhancing) and others with negative (silencing) effects. Of course, which of these DNA regions are active in controlling a gene depends on which transcription factor(s) are present in the nucleus. Sets of positive regulators will work together to coordinate and maximize gene expression when needed, and sets of negative regulators will bind negative regulatory elements to silence a gene.

We saw that in eukaryotes, the initiation of transcription involves many transcription factors and RNA polymerase II acting at a gene promoter to form a transcription preinitiation complex. TFIID, or TATA binding protein is one of the first factors to bend, causing the DNA in the promoter region to bend, much like the CAP protein in bacteria. TFIID also recruits other transcription factors to the promoter. As in bacteria, bending the DNA loosens H-bonds between bases, facilitating unwinding the double helix near the gene. Bending eukaryotic DNA also brings distal regulatory proteins bound to enhancer sequences far from the promoter together with the proteins bound to more proximal regulatory elements, as shown in the drawing below.

Nucleotide methyation sites may facilitate regulatory protein-enhancer binding. When such regulatory proteins, here called activators (i.e., of transcription), bind to their enhancers, they acquire an affinity for protein cofactors that enable recognition and binding to other proteins in the transcription initiation complex. This attraction stabilizes the bend in the DNA that then makes it easier for RNA polymerase II to initiate transcription

It is worth reminding ourselves that it is shape and allosteric change that allow DNAprotein interactions (in fact, any interactions of macromolecules). The lac repressor we saw earlier is a transcription factor with helix-turn-helix DNA binding motifs. This motif and two others (zinc finger, and leucine zipper) characterize DNA binding proteins are illustrated below.

DNA-binding motifs in each regulatory protein shown here bind one or more regulatory elements &lsquovisible&rsquo to the transcription factor in the major groove of the double helix.

We will look next at some common ways in which eukaryotic cells are signaled to turn genes on or off, or to increase or decrease their rates of transcription. As we describe these models, remember that eukaryotic cells regulate gene expression in response to changes in extracellular environments. These can be unscheduled, unpredictable changes in blood or extracellular fluid composition (ions, small metabolites), or dictated by changes in a long-term genetic program of differentiation and development. Changes in gene expression even obey circadian (daily) rhythms, the ticking of a clock. In eukaryotes, changes in gene expression, expected or not, are usually mediated by the timely release of chemical signals from specialized cells (e.g., hormones, cytokines, growth factors, etc.). We will focus on some betterunderstood models of gene regulation by these chemical signals.

DNA Transcription + Translation + Regulation of Gene Expression

Exons are the protein coding regions of an mRNA. All mRNAs code for proteins and so all mRNAs contain exons. In contrast, functional RNAs such as tRNAs and mRNAs do not code for proteins and so do not contain exons

Introns - intervening sequences (don't code for proteins/functional RNAs)

2. Free end of 5' splice site (exon 1) reacts w/3' splice site (exon 2) → linking the two exons together covalently and cutting out the intron

binds mRNA at 5' splice site, 3' splice site, and branch point A

ex. can make muscle proteins with slightly different functions for each muscle (striated, smooth, brain, etc.)

5' cap consists of adding 7' methyl guanosine stabilizes transcription and assists in nuclear export

RNA w/ 5', 3' modifications - exported from the nucleus to the cytoplasm where they are translated

Groups of 3 nucleotides = codons → codes for 1 amino acid

64 possible codons but only 20 amino acids

Nonsense - aa changed to a stop codon

A. Deletion of four consecutive nucleotides in the middle of the coding sequence

B. insertion of a single nucleotide near the end of the coding sequence

C. Deletion of three consecutive nucleotides in the middle of the coding sequence

Untranslated regions (UTR) - parts of mRNA not translated into proteins

5' Untranslated Regions (5' UTRs) are sequences after the +1 nucleotide but before the first AUG.

3' UTRs are sequences between the stop codon and the polyA tail.

Gene = 1 ORF/mRNA - eukaryotes have this

Anticodon (one end of tRNA) - 3 nucleotides base pair with codon in mRNA

3' amino acid acceptor stem = 3' end of tRNA where amino acid is covalently attached

3rd nucleotide in the codon is in the wobble position (base pairing is more flexible so you can form some abnormal base pairs)

P-site: binds peptidyl tRNA

-----> Incorrectly base-paired tRNAs: dissociate

Release factor (protein) = binds A site and interacts w/stop codon w/amino acids instead of anticodon

Release factor has a similar structure to a tRNA

Release factor (protein) = binds A site and interacts w/stop codon w/amino acids instead of anticodon

Release factor has a similar structure to a tRNA

Release factor (protein) = binds A site and interacts w/stop codon w/amino acids instead of anticodon

Release factor has a similar structure to a tRNA

Once the mRNA is in the cytoplasm, it can be packed with many ribosomes simultaneously translating it into a protein

Polysome (prokaryotes and eukaryotes): 1 mRNA and lots of ribosomes

--> Allow rapid translation of each mRNA

most often used b/c it's the most efficient in terms of energy

Protein regulation: e.g. phosphorylating the protein

Uses more energy because you've translated a protein you might not use

Basal transcription level: default level of transcription w/out adjustment

Specific transcription factors (TF): unlike general transcription factors, are necessary for the transcription of only one or a few genes

Change the transcription level above or below the basal level:

Repressor: decrease transcription

Activator: increase transcription

Trp operon - Trp synthesis enzyme

Sequence in the promoter that binds repressor protein (repressor not coded for in Trp operon)

In the presence of Trp in the environment, repressor binds DNA and blocks transcription (by preventing RNA polymerase binding)

In the absence of Trp in environment, repressor won't bind DNA, so transcription is allowed

Trp operon - Trp synthesis enzyme

Sequence in the promoter that binds repressor protein (repressor not coded for in Trp operon)

In the presence of Trp in the environment, repressor binds DNA and blocks transcription (by preventing RNA polymerase binding)

In the absence of Trp in environment, repressor won't bind DNA, so transcription is allowed

Trp operon - Trp synthesis enzyme

Sequence in the promoter that binds repressor protein (repressor not coded for in Trp operon)

In the presence of Trp in the environment, repressor binds DNA and blocks transcription (by preventing RNA polymerase binding)

In the absence of Trp in environment, repressor won't bind DNA, so transcription is allowed

Bacteria only transcribe lac operon if

Protein named lac permease

Protein named B-galactosidase

Inducer: small molecule that increases transcription - in this case, allolactose is the inducer (binds the LacI repressor)

Is constitutively (always) transcribed/expressed

So the presence of cAMP indicates the absence of glucose

cAMP allosterically binds CAP activator helping the RNA polymerase bind to the DNA, increasing transcription of Lac Operon

The simplest interpretation is
that the Lacheinmal gene contains a 173-nucleotide-long exon, and that this exon is lost during the processing of the mutant precursor mRNA (pre-mRNA). This could occur, for example, if the mutation changed the 3ʹ splice site in the preceding intron ("I1") so that it was no longer recognized by the splicing machinery (a change in the CAG sequence shown in Figure 7-19 could do this). The snRNP would search for the next available 3ʹ splice site, which is found at the 3ʹ end of the next intron ("I2"), and the splicing reaction would therefore remove E2 together with I1 and I2, resulting in a shortened mRNA. The mRNA is then translated into a defective protein, resulting in the Lacheinmal deficiency.

B. Correct. The aminoacyl-tRNA enters the ribosome at the A site and forms hydrogen bonds with the codon in the mRNA.

A second mutation in the cell's DNA leads to a single nucleotide change in a trNA that allows the correct translation of the protein that is, the second mutation "suppresses" the defect caused by the first. the altered trNA translates the ugA as tryptophan.

What nucleotide change has probably occurred in the mutant trNA molecule?

Many other protein-encoding sequences, however, contain UGA codons as their natural stop sites, and these stops would also be affected by the mutant tRNA.

Depending on the competition between the altered tRNA and the normal translation release factors, some of these proteins would be made with additional amino acids at their C-terminal end.

The 5' end of a gene is defined as the part that codes for the 5' end of the RNA. So the promoter (to the right) is at the 5' end of the gene, while transcription terminator (to the left) is towards the 3' end of the gene.

DNA binding domain binds the enhancer

Only one or a few genes have these enhancer sequences which is why the regulatory proteins are specific transcription factors

Directly interact with initiation complex (transcription complex) to help it assemble

Mediator acts as a link b/t specific and general transcription factors → helps transcription complex assemble

DNA methylation IS NOT histone modification --> this covalent modification generally turns off genes by attracting proteins that bind to methylated C's and block gene transcription

Proteins bind methylated 5' CG 3' (literally just add a methyl group coming off of the C on two CG/GC segments of antiparallel strands --> see notebook 10/9) and block transcription

Enzymes recognize if there's a methyl on old DNA strand and if there is they add a methyl to the newly synthesized strand

Self-regulated activators: proteins that increase their own transcription → affect multiple genes including themselves

Produces a positive feedback loop

Hormone turns on transcription of transcription factor leading to initial self-regulated activator expression
--> but once we have some activator, it increases its own transcription even after the hormone goes away creating a positive feedback loop

Self-regulated activators: proteins that increase their own transcription → affect multiple genes including themselves

Produces a positive feedback loop

Hormone turns on transcription of transcription factor leading to initial self-regulated activator expression
--> but once we have some activator, it increases its own transcription even after the hormone goes away creating a positive feedback loop

mRNA degraded if it loses 5' cap or 3' poly A tail

mRNA degraded if it loses 5' cap or 3' poly A tail

Complementary to specific mRNA
--> Bind the complementary mRNAs and either block translation or target the mRNA to be destroyed

Use RISC (RNA induced silencing complex) complex to do this

1 miRNA can regulate many genes because they can bind to the UTR of many mRNAs

This precursor miRNA is cut up to make a short double-stranded miRNA

Allosteric regulation (ex. GTP-binding)

proteasome = (large protein complex in cytoplasm): Degrades proteins back to amino acids

proteasome = (large protein complex in cytoplasm): Degrades proteins back to amino acids

Determine which of the following can explain why no enzyme Q activity is present even though lots of Q mRNA is present? Briefly explain your reasoning.

A) Enzyme Q is rapidly ubiquitinated.

B) Enzyme Q is activated by an allosteric regulator and this allosteric regulator is not present.

Lactose (or allolactose) binds to the lac repressor, reducing the affinity of the lac repressor to the
operator. This decreased affinity results in the promoter being accessible to RNA polymerase.

The lack of glucose allows for increased synthesis of cAMP, which can complex with CAP. The
formation of CAP-cAMP complexes improves the efficiency of RNA polymerase binding to the
promoter, which results in higher levels of transcription from the lac operon.

Condition 2 will result in the production of the least amount of β-galactosidase.* With no lactose
present, the lac repressor is active and binds to the operator, inhibiting transcription.

indicate how, through such contacts, a protein can distinguish a t-A from a c-g pair.

A. in bacteria, but not in eukaryotes, many mrnAs contain the coding region for more than one gene.

B. most dnA-binding proteins bind to the major groove of the dnA double helix.

Inducers function by disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes.

b) Cells express a mutant form of the tryptophan repressor that cannot bind to DNA?

c) Cells express a mutant form of the tryptophan repressor that cannot bind tryptophan?

d) The Trp repressor binds to DNA even in the absence of tryptophan?

Answer options:
1. Transcription would occur in the absence but not the presence of tryptophan.

2. Transcription would occur in the presence but not the absence of tryptophan.

3. Transcription would be constitutive

a) You only need to synthesize more tryptophan when it is absent from the cell, so the enzymes that synthesize Trp are only expressed in the absence of Trp.

b) The Trp repressor blocks transcription when it is bound to the operator region. If it cannot bind the DNA, it will never repress transcription and the genes will always be on.

c) The repressor protein normally only binds to the DNA when it is bound to tryptophan. If the protein cannot bind the Trp, the protein will never bind DNA and will never block transcription and the genes will always be on.

AraC is a transcription regulator that binds adjacent to the promoter of the arabinose operon. To understand the regulatory properties of the AraC protein, you can engineer mutant araC- bacteria in which the araC gene has been deleted and there is no functional AraC protein in the cell. You can then look at what effect the presence or absence of the AraC protein has on the arabinose operon expression levels.

(Answers can be used once, more than once, or not at all.)

1) Would you expect the arabinose operon to be expressed at high or low levels in WT cells in the presence of arabinose?

2) Would you expect the arabinose operon to be expressed at high or low levels in WT cells in the absence of arabinose?

3) If AraC worked as a gene repressor, would you expect the arabinose operon to be expressed at high or low levels in the presence of arabinose in the araC- mutant cells?

4) If the AraC protein worked as a gene repressor, would you expect the arabinose operon to be expressed at high or low levels in the absence of arabinose in the araC- mutant cells?

5) Your findings from the real experiment are summarized in the table below.

AraC - arabinose operon expression is low in both the presence and absence of arabinose

Do the results in the table indicate that the AraC protein regulates arabinose metabolism by acting as a gene repressor or a gene activator?

1 + 2) Since this operon codes for the enzymes responsible for metabolizing the sugar arabinose, the enzymes are needed when the sugar is present but they are not needed when the sugar is absent.

3 + 4) If the AraC protein acts as a gene repressor for the arabinose operon, the arabinose operon should be expressed at high levels in the presence or absence of arabinose when there is no AraC protein around. In fact, the the arabinose operon expression levels should be high all the time, regardless of the presence or absence of arabinose, because it should be transcribed under all conditions in the absence of AraC.

Reporter Gene Vectors

Vectors are plasmids, extrachromosomal elements, that carry reporter gene sequences for delivery into cultured cells. Not only do vectors provide the reporter gene, but may also have multiple cloning sites, promoters, response elements, polyadenylation sequences, mammalian selectable markers or other sequence elements that help with expression and selection. Reporter vectors are critical for success in reporter assays.

PGL4 Luciferase Reporter Vectors Encoding Firefly and Renilla Luciferase

Because vectors are used to deliver the reporter gene to host cells, regulatory sequences such as transcription factor-binding sites and promoter modules within the vector backbone can lead to high background and anomalous responses. This is a common issue for mammalian reporter vectors, including the pGL3 Luciferase Reporter Vectors. Our scientists applied the successful "cleaning" strategy described for reporter genes to the entire pGL3 Vector backbone, removing cryptic regulatory sequences wherever possible, while maintaining reporter functionality. In addition, the multiple cloning region was redesigned with a unique SfiI site for easy transfer of the DNA element of interest, the f1 origin of replication removed and an intron deleted. Furthermore, a synthetic poly(A) signal/transcriptional pause site was placed upstream of either the multiple cloning region (in promoterless vectors) or HSV-TK, CMV or SV40 promoter (in promoter-containing vectors). This extensive effort resulted in the totally redesigned and unique vector backbone of the pGL4 Vectors. There are also a series of pGL4 Vectors with partial deletions of the CMV promoter for nuanced usage of the strong promoter.

The pGL4 family of luciferase vectors incorporates a variety of features such as your choice of optimized firefly or Renilla luciferase genes, Rapid Response™ versions for improved temporal response, mammalian selectable markers, basic vectors without promoters, promoter-containing control vectors and predesigned vectors with your choice of several response elements (Figure 6).

Figure 6. The family of pGL4 Luciferase Reporter Vectors incorporates a variety of additional features, such as a choice of luciferase genes, Rapid Response&trade versions, a variety of mammalian selectable markers and vectors with or without promoters and response elements.

PNL Vectors Encoding NanoLuc® Luciferase

NanoLuc® luciferase is available in a variety of vectors for use in reporter gene assays of transcriptional control. These pNL vectors are based on the pGL4 vector backbone and thus offer many of the same advantages: Removal of transcription factor-binding sites and other potential regulatory elements to reduce the risk of anomalous results, easy sequence transfer from existing plasmids and a choice of several promoter sequences, including no promoter, minimal promoter and viral promoters. The family of vectors offer a choice of NanoLuc® genes (unfused Nluc, PEST-destabilized NlucP and secreted secNluc). These NanoLuc® gene variations are codon-optimized and have had many potential regulatory elements or other undesirable features such as common restriction enzyme sites removed.

In addition, there are coincidence reporter vectors that encode both NanoLuc® and firefly luciferases on a single transcript. These vectors come with no promoter, a minimal promoter or CMV promoter for use in high-throughput screening. Study the rate of protein turnover of two key signaling proteins involved in response to cellular stress using reporter vectors.

Reporter Vectors

Our extensive line of state-of-the-art bioluminescent reporter vectors includes the pGL4 Vectors —the latest generation of firefly and Renilla luciferase reporter vectors, and the pNL Vectors, which contain the NanoLuc® luciferase reporter gene.

Regulation of Gene Expression | Genetics

In this article we will discuss about the regulation of gene expression in prokaryotes and eukaryotes.

The DNA of a microbial cell consists of genes, a few to thousands, which do not express at the same time. At a particular time only a few genes express and synthesize the desired protein. The other genes remain silent at this moment and express when required. Requirement of gene expression is governed by the environment in which they grow. This shows that the genes have a property to switch on and switch off.

The Genetic Code that 20 different amino acids constitute different protein. All are synthesised by codons. Therefore, synthesis of all the amino acids requires energy which is useless because all the amino acids constituting proteins are not needed at a time.

Hence, there is need to control the synthesis of those amino acids (proteins) which are not required. By doing this the energy of a living cell is conserved and cells become more competent. Therefore, a control system is operative which is known as gene regulation.

There are certain substrates called inducers that induce the enzyme synthesis. For example, if yeast cells are grown in medium containing lactose, an enzyme lactase is formed. Lactase hydrolyses the lactose into glucose and galactose. In the absence of lactase, lactose synthesis does not occur.

This shows that lactose induces the enzyme lactase. Therefore, lactase is known as inducible enzyme. In addition, sometimes the end product of metabolism has inhibitory effect on the synthesis of enzyme. This phenomenon is called feed back or end product inhibition.

From the outgoing discussion it appears that a cell has auto-control mediated by the gene itself. For the first time Francois Jacob and Jacques Monod (1961) at the Pasteur Institute (Paris) put forward a hypothesis to explain the induction and repression of enzyme synthesis.

They investigated the regulation of activities of genes which controls lactose fermentation in E. coli through synthesis of an enzyme, β-galactosidase. For this significant contribution in the field of biochemistry they were awarded Nobel Prize in Medicine in 1965.

Regulation of Gene Expression in Prokaryotes:

Gene expression of prokaryotes is controlled basically at two levels i.e. transcription and translation stages. In addition, mRNA degradation and protein modification also play a role in regulation. Most of the prokaryotic genes that are regulated are controlled at transcriptional stage.

Other control measures operating at different levels are given in Table. 10.2:

Transcriptional Control in Prokaryotes:

It is a general strategy in a living organism that chemical changes occur by a metabolic pathway through a chain of reactions. Each step is determined by the enzymes. Again synthesis of an enzyme comes under the control of genetic material i.e. DNA in living organisms. Enzymes (proteins) are synthesised via two steps: transcription and translation.

Transcription refers to synthesis of mRNA. Transcription is regulated at or around promoter region of a gene. By controlling the ability of RNA polymerase to the promoter the cell can modulate the amount of message being transcribed through the structural gene. However, if RNA polymerase has bound, again it can modulate transcription.

By doing so the amount of gene product synthesized is also modulated. The coding region is also called structural gene. Adjacent to it are regulatory regions that control the structural genes. The regulatory regions are composed of promoter (for the initiation of transcription) and an operator (where a diffusible regulatory protein binds) regions.

The molecular mechanisms for each of regulatory patterns vary widely but usually fall in one of two major groups: negative regulation and positive regulation. In negative regulation an inhibitor is present in the cell and prevents transcription. This inhibitor is called as repressor.

An inducer i.e. antagonist repressor is required to permit the initiation of transcription. In a positive regulated system an effector molecule (i.e. a protein, molecule or molecular complex) activates a promoter. The repressor proteins produce negative control, whereas the activator proteins produce positive control.

Since the transcription process is accomplished in three steps (RNA polymerase binding, isomerization of a few nucleotides and release of RNA polymerase from promoter region), the negative regulators usually block the binding, whereas the activators interact with RNA polymerase making one or more steps.

Fig. 10.19 shows the negative and positive regulation mechanism of the genes. In negative regulation (A) an inhibitor is bound to the DNA molecule. It must be removed for efficient transcription. In positive regulation (B) an effector molecule must bind to DNA for transcription.

i. The Lac Operon Model (Jacob-Monod Model):

For the first time Jacob and Monod (1961) gave the concept of operon model to explain the regulation of gene action. An operon is defined as several distinct genes situated in tandem, all controlled by a common regulatory region.

Commonly an operon consists of repressor, promoter, operator and structural genes. The message produced by an operon is polycistronic because the information of all the structural genes resides on a single molecule of mRNA.

The regulatory mechanism of operon responsible for utilization of lactose as a carbon source is called the lac operon. It was extensively studied for the first time by Jacob and Monod (1961). Lactose is a disaccharide which is composed of glucose and galactose (Fig. 10.20).

The lactose utilizing system consists of two types of components the structural genes (lacZ, lacY and lacA) the products of which are required for transport and metabolism of lactose and the regulatory genes (the lad, the lacO and the lacP). These two components together comprises of the lac operon (Fig. 10.21a).

One of the most key features is that operon provides a mechanism for the coordinate expression of structural genes controlled by regulatory genes. Secondly, operon shows polarity i.e. the genes Z, Y and A synthesise equal quantities of three enzymes β-galactosidase (by lacZ), permease (by lacY) and acetylase (by lacA). These are synthesized in an order i.e. β- galactosidase first and acetylase in the last.

(i) The Structural Genes:

The structural genes form one long polycistronic mRNA molecule. The number of structural gene corresponds to the number of proteins. Each structural gene is controlled independently, and transcribes mRNA molecules separately.

This depends on substrates to be utilized. For example, in lac operon three structural genes (Z, Y and A) are associated with lactose utilization (Fig. 10.21A). β-galactose is the product of lacZ that cleaves β-1 → 4 linkage of lactose and releases the free monosaccharides.

This enzyme is a tetramer of four identical subunits each with molecular weight of 1,16,400. The enzyme permease (a product of lacY) facilitates the lactose to enter inside the bacterium.

Permease has molecular weight of 46,500. It is hydrophobic. The cells mutant in lacZ and lacY are designated as Lac – i.e. the bacteria cannot grow in lactose-free medium. The enzyme transacetylase (30,000 MW) is a product of lacA whose no definite role has been assigned.

The lac operon consists of a promoter (P) and an operator (O) together with the structural genes. The initiation codon of lacZ is TAG that corresponds to AUG of mRNA. It is situated 10 bp away from the end of operator gene. However, the lac operon cannot function in the presence of sugars other than lactose.

The operator gene is about 28 bp in length present adjacent to lacZ gene. The base pairs in the operator region are palindrome i.e. show two fold symmetry from a point (Fig. 10.22). The operator overlaps the promoter region.

The lac repressor proteins (a tetramer of four subunits) bind to the lac operator in vitro and protect part of the lac operator in vitro and protect part of the promoter region from the digestion of DNase.

The repressor proteins bind to the operator and form an operator-repressor complex which in turn physically blocks the transcription of Z,Y and A genes by preventing the release of RNA polymerase to begin transcription (Fig. 10.21b).

In bacteriophage λ there are two operators the OL and OR which have different base sequences. Lambda repressor (gpcl) is rapidly synthesized, binds to OL and OR and inhibits the synthesis of mRNA and production of proteins gpcll and gpcII.

(iii) The Promoter Gene:

The promoter gene is about 100, nucleotide long and continuous with the operator gene. Gilbert (1974) and Dickson (1975) have worked out the complete nucleotide sequence of the control region of lac operon. The promoter gene lies between the operator gene and regulator gene.

Like operators the promoter region consists of palindromic sequence of nucleotides (Figs. 10.22 and 10.23). These palindromic sequences are recognized by such proteins that have symmetrically arranged subunits. This section of two fold symmetry is present on the CRP site that binds to a protein called CRP (cyclic AMP receptor protein). The CRP is encoded by CRP gene (Fig. 10.25).

It has been shown experimentally that CRP binds to cAMP (cyclic AMP found in E. coli and other organisms) molecule and form a cAMP-CRP complex. This complex is required for transcription because it binds to promoter and enhances the attachment of RNA polymerase to the promoter.

Therefore, it increases transcription and translation processes. Thus, cAMP-CRP is a positive regulator in contrast to the repressor, and the lac operon is controlled by both positively and negatively.

According to a model proposed by Pribnow (1975) the promoter region consists of three important components which are present at a fixed position to each other.

These components are:

(i) The recognition sequence,

(ii) The binding sequence, and

(iii) An mRNA initiation site.

The recognition sequence is situated outside the polymerase binding site that is why it is protected from DNase. Firstly, RNA polymerase binds to DNA and forms a complex with the recognition sequence. The binding site is 7 bp long (5’TATGTTG) and present at such region that is protected from DNase. In other organisms the base pairs do not differ from more than two bases. Hence, it can be written as 5′ TATPuATG.

The mRNA initiation site is present near the binding site on one of the two bases. The initiation site is also protected from DNase. However, there is overlapping of promoter and operator in lac operon, Moreover, there is a sequence 5’CCGG, 20 bp left to mRNA initiation site. This is known as Hpall site (5’CCGG) because of being cleaved at this site by the restriction enzyme Hpall.

(iv) The Repressor (Regulator) Gene:

Repressor gene determines the transcription of structural gene.

It is of two types:

It codes for amino acid of a defined repressor protein.

After synthesis the repressor molecules are diffused from the ribosome and bind to the operator in the absence of an inducer. Finally, the path of RNA polymerase is blocked and mRNA is not transcribed. Consequently, no protein synthesis occurs. This type of mechanism occurs in the inducible system of active repressor.

Moreover, when an inducer (e.g. lactose) is present, it binds to repressor proteins and forms an inducer-repressor complex. This complex cannot bind to the operator. Due to formation of complex the repressor undergoes changes in conformation of shape and becomes inactive. Consequently, the structural genes can synthesise the polycistronic mRNAs and the later synthesizes enzymes (proteins).

In contrast, in the reversible system the regulator gene synthesizes repressor protein that is inactive and, therefore, fails to bind to operator. Consequently, proteins are synthesised by the structural genes.

However, the repressor proteins can be activated in the presence of a co-repressor. The co-repressor together with repressor proteins forms the repressor-co-repressor complex. This complex binds to operator gene and blocks protein synthesis.

Jacob and Monod (1961) could not identify the repressor protein. Gilbert and Muller – Hill (1966) succeeded in isolating the lac repressor from the Lac mutant cells of E. coli inside which the lac repressor was about ten times greater than the normal cells. The lac repressor proteins have been crystallized. It has a molecular weight of about 1,50,000.

It consists of four subunits-each has 347 amino acid residues and molecular weight of about 40,000 Daltons. The repressor proteins have strong affinity for a segment of 12-15 base pairs of operator gene. This binding of repressor blocks the synthesis of mRNA transcript by RNA polymerase.

The lac operon is induced when E. coli cells are kept in medium containing lactose. The lactose is taken up inside the cell where it undergoes glycosylation i.e. molecular rearrangement from lactose to allolactose. The galactosyl residue is present on 6 rather than 4 position of glucose (Fig. 10.20). Glycosylation is done by β-galactosidase that is constitutively present in the cell before induction.

Allolactose is the real inducer molecule. The lac repressor protein is an allosteric molecule with specific binding sites for DNA and inducer. Allolacctose binds to lac repressor to form an inducer- repressor complex. Binding of inducer to repressor allosterically changes the repressor lowering its affinity for lacO DNA.

Consequently repressor is released from lacO due to changes in three dimensional conformations. This is called allosteric effect. After being free lacO allows the RNA polymerase to form mRNA transcript. Here, allolactose acts as the effector molecule and checks the regulatory protein from binding to lacO (operator) gene.

ii. Positive Regulation of the lac Operon-Catabolic Control:

Cyclic AMP (cAMP) is the small molecule which is distributed in animal tissues, and controls the action of many hormones. It is also present in E. coli and the other bacteria. The cAMP is synthesized by the enzyme adenyl cyclase. (Fig. 10.24). Its concentration is directly regulated by glucose metabolism.

The Lac operon has an additional positive regulatory control mechanism to avoid the wastage of energy during the synthesis of lactose-utilizing proteins while there is adequate supply of glucose.

When E. coli grows in a medium containing glucose the cAMP concentration in the cells falls down. This mechanism is poorly understood. However, the note worthy point is that cAMP regulates the activity of lac operon (and other operons also).

In contrast when E. coli cells are fed with alternate carbon source e.g. succinate, cAMP level increases. The crp locus expresses the enzyme adenylate cyclase that converts the ATP to cAMP.

How does cAMP increase the process of transcription, is not known clearly. It has been shown experimentally that cAMP binds to the proteins expressed by crp locus which is known as cAMP receptor protein (CRP) or catabolic activator protein (CAP) (Fig. 10.25).

Therefore, CRP-cAMP complex binds to the CAP-binding site present on lac promoter. The CRP -cAMP bound complex promotes the helix destabilization downstream, and facilitates RNA polymerase binding. This results in efficient open promoter formation and in turn transcription.

iii. The PaJaMo Experiment:

The key experiment in understanding the induction of β-galactosidase was done by Arthur Pardee, Jacob and Monod therefore, it is called PaJaMo experiment. They found that if a DNA molecule containing the lac operon enters a cell devoid of lac operon (lac – ), then the lac – cells are converted in to lac + cells.

The lac operon expresses in the new cells, provided the DNA contains complete genes or open reading frames and a good promoter. The genes express and RNA polymerase binds to the promoter. The genes are transcribed, ribosomes bind to the mRNA, and β-galactosidase is synthesised.

II. Regulation of Gene Expression in Eukaryotes:

There is much variation and complexity in regulation of genes in eukaryotes. Because in eukaryotes different genes are expressed at different developmental stages of cells or different tissues under the influence of different types of stimuli imposed by external environment. Eukaryotic DNA undergoes several changes such as double stranded, linear thread, nucleosome, fibres, chromatid and chromosomes.

Gene expression and regulation take place only when DNA is in double stranded linear form. Moreover, if the promoter or regulator region of any gene is organized into chromosome, initiation of transcription does not take place.

Therefore, changes in state of chromatin occur by chromatin remodeling which results in gene activation. Thus packaging of DNA influences gene expression. In majority of cases regulation of gene expression takes place at transcription level. Regulation of expression at processing or translation level may also occur in eukaryotes.

Gene expression can be regulated at several steps in the pathway from DNA to RNA to protein in a cell as described below:

i. Transcriptional control:

Controlling the gene expression during transcription

ii. RNA processing control:

Control 8f processing of primary RNA transcripts to form mature mRNA

iii. RNA transport control:

Control of transport of mature mRNA from nucleus to cytoplasm

iv. Translational control:

Selection of mRNAs in cytoplasm to be translated by ribosome.

v. mRNA degradation control:

Selective degradation of certain mRNA molecules in the cytoplasm, or

vi. Protein activity control:

Selective activation, inactivation or compartmentalization of specific protein molecule after their synthesis. Only transcriptional control ensures that no superfluous intermediates are synthesized.

(i) Regulation through Transcriptional Factors:

Unlike prokaryotes, there are multiple DNA binding proteins called transcription factors that control transcription in eukaryotes. These proteins are grouped into two major classes: the general transcriptional factors (GTFs) and the regulatory transcriptional factors (RTFs) The eukaryotic RNA polymerase fails to recognize the promoter directly.

Therefore, the GTFs bind first the promoter directly (TATA sequence of all prokaryotes). RNA polymerase starts transcription at promoter site. The RTFs bind the regulatory site of the genes which is far away from the promoter.

The RTFs bind to all the regulatory sequences of gene and control the rate of assembly of GTFs at the promoter. The RTFs either increase or decrease the transcription. When transcription is increased, this property is called activator. The decreasing level of transcription is called repression.

(ii) Britten-Davidson Model for Gene Regulation:

Regulation at transcription level involves both activation and repression of genes. Because genes may be switched on in some cases and switched off in others. Various models have been proposed for regulation of gene expression in eukaryotes. In 1969, Britten and Davidson proposed a model called gene battery model or Britten- Davidson model which is very popular. This model was further elaborated in 1973.

According to this model, there are four classes of sequences:

(i) Producer genes (which are comparable to structural genes of prokaryotes),

(ii) Receptor site (comparable to operator gene in bacterial operon),

(iii) Integrator gene (comparable to regulator gene synthesizing an activator RNA which may or may not synthesize protein before it activates the receptor site), and

(iv) Sensor site (regulates the activity of integrator gene which can be transcribed only after activation of sensor site). The four classes of se­quences are interrelated (Fig. 10.27).

In this model producer gene and integrator gene are involved in transcription, whereas the receptor and sen­sor sequences help in recogni­tion without participating in RNA synthesis.

It has been proposed that receptor site and integrator gene are repeated several times so that the activ­ity of a large number of genes may be controlled in the same cell, same activator may rec­ognize all the repeats, and sev­eral enzymes of one pathway may be synthesized simultaneously.

Transcription of the same gene is done in different developmental stages. This is achieved by several receptor sites and integrator genes. Each producer gene possesses many receptors sites, each site responds to one activator (Fig. 10.28) so that several genes can be recognized by a single activator. But at different time the same gene may be activated by different activators.

A set of structural genes controlled by one sensor site is called ‘gene battery’. Several sets of genes may be activated when major changes are required. If one sensor site gets associated with them, transcription of all integrators may be caused at the same time. Thus, transcription of several producer genes is caused through receptor sites.


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Where to put the gene after eukaryotic promoter for best expression levels? - Biology

To understand how gene expression is regulated, we must first understand how a gene codes for a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different manners.

Prokaryotic organisms are single-celled organisms that lack a cell nucleus, and their DNA therefore floats freely in the cell cytoplasm. To synthesize a protein, the processes of transcription and translation occur almost simultaneously. When the resulting protein is no longer needed, transcription stops. As a result, the primary method to control what type of protein and how much of each protein is expressed in a prokaryotic cell is the regulation of DNA transcription. All of the subsequent steps occur automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level.

Eukaryotic cells, in contrast, have intracellular organelles that add to their complexity. In eukaryotic cells, the DNA is contained inside the cell’s nucleus and there it is transcribed into RNA. The newly synthesized RNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the RNA into protein. The processes of transcription and translation are physically separated by the nuclear membrane transcription occurs only within the nucleus, and translation occurs only outside the nucleus in the cytoplasm. The regulation of gene expression can occur at all stages of the process (Figure 1). Regulation may occur when the DNA is uncoiled and loosened from nucleosomes to bind transcription factors (epigenetic level), when the RNA is transcribed (transcriptional level), when the RNA is processed and exported to the cytoplasm after it is transcribed (post-transcriptional level), when the RNA is translated into protein (translational level), or after the protein has been made (post-translational level).

Figure 1. Prokaryotic transcription and translation occur simultaneously in the cytoplasm, and regulation occurs at the transcriptional level. Eukaryotic gene expression is regulated during transcription and RNA processing, which take place in the nucleus, and during protein translation, which takes place in the cytoplasm. Further regulation may occur through post-translational modifications of proteins.

The differences in the regulation of gene expression between prokaryotes and eukaryotes are summarized in Table 1. The regulation of gene expression is discussed in detail in subsequent modules.

Table 1. Differences in the Regulation of Gene Expression of Prokaryotic and Eukaryotic Organisms
Prokaryotic organisms Eukaryotic organisms
Lack nucleus Contain nucleus
DNA is found in the cytoplasm DNA is confined to the nuclear compartment
RNA transcription and protein formation occur almost simultaneously RNA transcription occurs prior to protein formation, and it takes place in the nucleus. Translation of RNA to protein occurs in the cytoplasm.
Gene expression is regulated primarily at the transcriptional level Gene expression is regulated at many levels (epigenetic, transcriptional, nuclear shuttling, post-transcriptional, translational, and post-translational)

Evolution of Gene Regulation

Prokaryotic cells can only regulate gene expression by controlling the amount of transcription. As eukaryotic cells evolved, the complexity of the control of gene expression increased. For example, with the evolution of eukaryotic cells came compartmentalization of important cellular components and cellular processes. A nuclear region that contains the DNA was formed. Transcription and translation were physically separated into two different cellular compartments. It therefore became possible to control gene expression by regulating transcription in the nucleus, and also by controlling the RNA levels and protein translation present outside the nucleus.

Some cellular processes arose from the need of the organism to defend itself. Cellular processes such as gene silencing developed to protect the cell from viral or parasitic infections. If the cell could quickly shut off gene expression for a short period of time, it would be able to survive an infection when other organisms could not. Therefore, the organism evolved a new process that helped it survive, and it was able to pass this new development to offspring.

  • Unique introduction to synthetic biology
  • Hands-on exploration of the role of promoters in gene regulation
  • Seamlessly blends together multiple areas of biology
  • Takes a traditional transformation lab to the next level
  • Lesson can be facilitated in 6 lab sessions, at most
  • Bring the unique area of synthetic biology into your classroom with this one-of-a-kind kit activity. Your students get the opportunity to take on the role of genetic engineers as they clone promoters into 2 different plasmids. This hands-on approach to transcription regulation exposes your class to a wide array of biological disciplines including biotechnology, molecular biology, and evolutionary biology.

Students clone the promoters into the plasmids, pClone Red and pClone Blue, using a common cloning technique, Golden Gate Assembly (GGA). After cloning the promoters, students are tasked with performing a transformation on E. coli cells to produce new ampicillin-resistant colonies expressing their designed plasmids. Based on the orientation of the promoter, students then observe colonies that appear green (produce GFP), red (produce RFP), or blue (produce AmilCP blue). This gives them 3 distinct colonies to further assess using ImageJ software, which is freely available, to determine the level of protein expression.

The kit includes enough materials for 8 groups of 2 to 4 students. The activities typically take 3 to 6 class periods for completion. Note: Order the kit with the perishable materials included or with a prepaid coupon to request perishables later at your convenience. Contact us or return the coupon to request delivery of the perishable materials.

28.2.4. The TATA-Box-Binding Protein Initiates the Assembly of the Active Transcription Complex

Cis-acting elements constitute only part of the puzzle of eukaryotic gene expression. Transcription factors that bind to these elements also are required. For example, RNA polymerase II is guided to the start site by a set of transcription factors known collectively as TFII (TF stands for transcription factor, and II refers to RNA polymerase II). Individual TFII factors are called TFIIA, TFIIB, and so on. Initiation begins with the binding of TFIID to the TATA box (Figure 28.19).

Figure 28.19

Transcription Initiation. Transcription factors TFIIA, B, D, E, and F are essential in initiating transcription by RNA polymerase II. The step-by-step assembly of these general transcription factors begins with the binding of TFIID (purple) to the TATA (more. )

The key initial event is the recognition of the TATA box by the TATA-box-binding protein (TBP), a 30-kd component of the 700-kd TFIID complex. TBP binds 10 5 times as tightly to the TATA box as to noncognate sequences the dissociation constant of the specific complex is approximately 1 nM. TBP is a saddle-shaped protein consisting of two similar domains (Section 7.3.3 Figure 28.20). The TATA box of DNA binds to the concave surface of TBP. This binding induces large conformational changes in the bound DNA. The double helix is substantially unwound to widen its minor groove, enabling it to make extensive contact with the antiparallel β strands on the concave side of TBP. Hydrophobic interactions are prominent at this interface. Four phenylalanine residues, for example, are intercalated between base pairs of the TATA box. The flexibility of AT-rich sequences is generally exploited here in bending the DNA. Immediately outside the TATA box, classical B-DNA resumes. This complex is distinctly asymmetric. The asymmetry is crucial for specifying a unique start site and ensuring that transcription proceeds unidirectionally.

Figure 28.20

Complex Formed by TATA-Box-Binding Protein and DNA. The saddlelike structure of the protein sits atop a DNA fragment that is both significantly unwound and bent.

TBP bound to the TATA box is the heart of the initiation complex (see Figure 28.19). The surface of the TBP saddle provides docking sites for the binding of other components (Figure 28.21). Additional transcription factors assemble on this nucleus in a defined sequence. TFIIA is recruited, followed by TFIIB and then TFIIF𠅊n ATP-dependent helicase that initially separates the DNA duplex for the polymerase. Finally, RNA polymerase II and then TFIIE join the other factors to form a complex called the basal transcription apparatus. Sometime in the formation of this complex, the carboxyl-terminal domain of the polymerase is phosphorylated on the serine and threonine residues, a process required for successful initiation. The importance of the carboxyl-terminal domain is highlighted by the finding that yeast containing mutant polymerase II with fewer than 10 repeats is not viable. Most of the factors are released before the polymerase leaves the promoter and can then participate in another round of initiation.

Figure 28.21

Assembly of the Initiation Complex. A ternary complex between the TATA-box-binding protein (purple), TFIIA (orange), and DNA. TFIIA interacts primarily with the other protein.

Although bacteria lack TBP, archaea utilize a TBP molecule that is structurally quite similar to the eukaryotic protein. In fact, transcriptional control processes in archaea are, in general, much more similar to those in eukaryotes than are the processes in bacteria. Many components of the eukaryotic transcriptional machinery evolved from an ancestor of archaea.

Pichia pastoris as an expression host for membrane protein structural biology

Pichia pastoris is a well established recombinant expression system.

A number of integral membrane proteins have been produced in this system.

The protein has been used to solve several high resolution structures.

Key features of the P. pastoris expression system are described.

A summary of the tools available for membrane protein expression is included.

The methylotrophic yeast Pichia pastoris is a widely used recombinant expression host. P. pastoris combines the advantages of ease of use, relatively rapid expression times and low cost with eukaryotic co-translational and post-translational processing systems and lipid composition. The suitability of P. pastoris for high density controlled culture in bioreactors means large amounts of protein can be obtained from small culture volumes. This review details the key features of P. pastoris, which have made it a particularly useful system for the production of membrane proteins, including receptors, channels and transporters, for structural studies. In addition, this review provides an overview of all the constructs and cell strains used to produce membrane proteins, which have yielded high resolution structures.

Watch the video: Molecular Biology Session 16 Regulation of Gene Expression p1 (May 2022).