Glycogen Biosynthesis

Biosynthetic and degradative pathways rarely operate by precisely the same reactions in the forward and reverse directions. Glycogen metabolism provided the first known example of this important principle. Separate pathways afford much greater flexibility, both in energetics and in control. In 1957, Luis Leloir and his coworkers showed that glycogen is synthesized by a pathway that utilizes uridine diphosphate glucose (UDP-glucose) rather than glucose 1-phosphate as the activated glucose donor.

Step 1. UDP-Glucose Is an Activated Form of Glucose

 
UDP-glucose, the glucose donor in the biosynthesis of glycogen, is an activated form of glucose, just as ATP and acetyl CoA are activated forms of orthophosphate and acetate, respectively. The C-1 carbon atom of the glucosyl unit of UDPglucose is activated because its hydroxyl group is esterified to the diphosphate moiety of UDP. UDP-glucose is synthesized from glucose 1-phosphate and uridine triphosphate (UTP) in a reaction catalyzed by UDPglucose pyrophosphorylase.




 The pyrophosphate liberated in this reaction comes from the outer two phosphoryl residues of UTP. This reaction is readily reversible. However, pyrophosphate is rapidly hydrolyzed in vivo to orthophosphate by an inorganic pyrophosphatase. The essentially irreversible hydrolysis of pyrophosphate drives the synthesis of UDP-glucose. The synthesis of UDP-glucose exemplifies another recurring theme in biochemistry: many biosynthetic reactions are driven by the hydrolysis of pyrophosphate.
Step 2. Glycogen Synthase Catalyzes the Transfer of Glucose from UDP-Glucose to a Growing Chain

New glucosyl units are added to the nonreducing terminal residues of glycogen. The activated glucosyl unit of UDPglucose is transferred to the hydroxyl group at a C-4 terminus of glycogen to form an a-1,4-glycosidic linkage. In elongation, UDP is displaced by the terminal hydroxyl group of the growing glycogen molecule. This reaction is catalyzed by glycogen synthase, the key regulatory enzyme in glycogen synthesis. Glycogen synthase can add glucosyl residues only if the polysaccharide chain already contains more than four residues.Thus, glycogen synthesis requires a primer. This priming function is carried out by glycogenin, a protein composed of two identical 37-kd subunits, each bearing an oligosaccharide of a-1,4-glucose units. Carbon 1 of the first unit of this chain, the reducing end, is covalently attached to the phenolic hydroxyl group of a specific tyrosine in each glycogenin subunit. How is this chain formed? Each subunit of glycogenin catalyzes the addition of eight glucose units to its partner in the glycogenin dimer. UDP-glucose is the donor in this autoglycosylation. At this point, glycogen synthase takes over to extend the glycogen molecule.

Step 3. A Branching Enzyme Forms a-1,6 Linkages

Glycogen synthase catalyzes only the synthesis of a-1,4 linkages. Another enzyme is required to form the a-1,6 linkages that make glycogen a branched polymer. Branching occurs after a number of glucosyl residues are joined in a-1,4 linkage by glycogen synthase. A branch is created by the breaking of an a-1,4 link and the formation of an a-1,6 link: this reaction is different from debranching. A block of residues, typically 7 in number, is transferred to a more interior site. The branching enzyme that catalyzes this reaction is quite exacting. The block of 7 or so residues must include the nonreducing terminus and come from a chain at least 11 residues long. In addition, the new branch point must be at least 4 residues away from a preexisting one. Branching is important because it increases the solubility of glycogen. Furthermore, branching creates a large number of terminal residues, the sites of action of glycogen phosphorylase and synthase. Thus, branching increases the rate of glycogen synthesis and degradation.Glycogen branching requires a single transferase activity. Glycogen debranching requires two enzyme activities: a transferase and an a-1,6 glucosidase. Sequence analysis suggests that the two transferases and, perhaps, the a-1,6 glucosidase are members of the same enzyme family, termed the a -amylase family. Such an enzyme catalyzes a reaction by forming a covalent intermediate attached to a conserved aspartate residue . Thus, the branching enzyme appears to function through the transfer of a chain of glucose molecules from an a-1,4 linkage to an aspartate residue on the enzyme and then from this site to a more interior location on the glycogen molecule to form an a-1,6 linkage.

Glycogen Synthase Is the Key Regulatory Enzyme in Glycogen Synthesis

The activity of glycogen synthase, like that of phosphorylase, is regulated by covalent modification. Glycogen synthase is phosphorylated at multiple sites by protein kinase A and several other kinases. The resulting alteration of the charges in the protein lead to its inactivation . Phosphorylation has opposite effects on the enzymatic activities of glycogen synthase and phosphorylase. Phosphorylation converts the active a form of the synthase into a usually inactive b form. The phosphorylated b form requires a high level of the allosteric activator glucose 6-phosphate for activity, whereas the a form is active whether or not glucose 6-phosphate is present. 
Glycogen Is an Efficient Storage Form of Glucose

What is the cost of converting glucose 6-phosphate into glycogen and back into glucose 6-phosphate? The pertinent reactions have already been described, except for reaction 5, which is the regeneration of UTP. ATP phosphorylates UDP in a reaction catalyzed by nucleoside diphosphokinase. Thus, one ATP is hydrolyed incorporating glucose 6-phosphate into glycogen. The energy yield from the breakdown of glycogen is highly efficient. About 90% of the residues are phosphorolytically cleaved to glucose 1-phosphate, which is converted at no cost into glucose 6-phosphate. The other 10% are branch residues, which are hydrolytically cleaved. One molecule of ATP is then used to phosphorylate each of these glucose molecules to glucose 6-phosphate. The complete oxidation of glucose 6-phosphate yields about 31 molecules of ATP, and storage consumes slightly more than one molecule of ATP per molecule of glucose 6-phosphate; so the overall efficiency of storage is nearly 97%

 

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SDS PAGE

Principle

Electrophoresis is the study of the movement of charged molecules in an electric field. The generally usedsupport medium is cellulose or thin gels made up of either polyacrylamide or agarose. Cellulose is used as support medium for low molecular weight biochemicals such as amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for larger molecules like proteins. The general electrophoresis techniques cannot be used to measure the molecular weight of the biologicalmolecules because the mobility of a substance in the gel is influenced by both charge and size. In order toovercome this, if the biological samples are treated so that they have a uniform charge, electrophoretic mobilitythen depends primarily on size. The molecular weight of protein maybe estimated if they are subjected to electrophoresis in the presence of a detergent sodium dodecyl sulfate (SDS) and a reducing agent
mercaptoethanol (b ME). SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptidechain coated with negatively charged SDS molecules. 1.4grams of SDS binds per gram of protein. Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.

SDS-Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross linking agent N N’ methylene bis acrylamide

Acrylamide + N N’ methylene bis acrylamide

Add Chemical Ammonium persulfate (catalyst)
+
Polymerisation ↓ TEMED (N,N N’ N’ tetramethylethylene diamine

↓

Polyacrylamide

Procedure:

1. Assembling the glass plate

( Gloves should be worn at all times while performing SDS-PAGE. To insure proper alignment and casting, the glass plates, spacers, combs and casting stand gaskets must be clean and dry. The glass plates should be cleaned with 70% ethanol.)

1. Assemble the glass plate on a clean surface. Lay the longer glass plate down first, then place two spacers of equal thickness along the rectangular plate. Next place the shorter glass plate on top of the spacers so that the bottom ends of the spacers and glass plates are aligned

2. Loosen the 4 screws on the clamp assembly and stand it up so that the screws are facing away from you. Firmly grasp the glass plate sandwich with the longer plate facing away from you, and gently slide it into the clamp assembly. Tighten the top 2 screws of the clamp assembly.

3. Place the clamp assembly into the alignment slot of the casting stand so that the clamp screws faceaway from you. Loosen the top 2 screws to allow the plates and spacers to sit firmly against the casting stand base. Gently tighten all the screws

4. Pull the completed sandwich from the alignment slot. Check that the plates and spacers are aligned. If not, realign the sandwich as in steps 1-3. Before transferring the clamp assembly to the casting slot,recheck the alignment of the spacers. Do this by inverting the gel sandwich and looking at the surface of the 2 glass plates and the spacer. Make sure that they are aligned.

5. Transfer the clamp assembly to one of the casting slots in the casting stand. If 2 gels are to be prepared, place the clamp assembly on the other side of the alignment slot.

6. Press the acrylic pressure plate bottom, so that the glass plates rest on the rubber gasket. Snap the acrylic plate underneath the overhang of the casting slot. Do not push the glass plates or spacers because this could break the glass plate.

2. Casting the gels (Demonstration)

Prepare 10% resolving/separating gel and 4.5% stacking gel.

1. Prepare the separating gel monomer solution by combining all reagents except ammonium persulfate(APS) and TEMED. Deaerate and mix the solution after adding each reagent by swirling the container
gently.

2. Place a comb completely into the assembled gel sandwich. With a marker pen, place a mark on the Glass plate 1 cm below the teeth of the comb. This will be the level to which the separating gel is poured. Remove the comb.

3. Add APS and TEMED to the monomer solution and mix well by swirling gently. Pipette the solution to the mark.

4. Immediately overlay the monomer solution with 1 ml. of water. Use a steady, even rate of delivery to prevent mixing with the gel.

5. Allow the gel to polymerize for 45 minutes to 1 hour. Pour the water overlaying the gel and drain the excess water with strips of filter paper.

6. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Deaerate and mix the solution by swirling gently.

7. Place a comb in the gel sandwich.

8. Add APS and TEMED to the solution and pipette the solution down one of the spacer until the sandwich is filled completely

9. Allow the gel to polymerize for 15 minutes.

10. Remove the comb.

11. Gel is placed in the buffer chamber and running gel buffer is added into the chamber

3. Preparation of samples. ( here a cloned protein is put under analysis)

From the recombinant clone VC-25, the recombinant protein is produced as follows:

The clone was grown for 4hours and induced using IPTG for next four hours. The culture was pelleted and resuspended in PBS

4. Loading the samples

1. Rinse the syringe to be used for loading samples a few times with distilled water. Demonstrators will load the first well with LMW (7 ml of LMW). Insert the syringe to about 1-2 mm from the well bottom before delivery. Rinse the syringe a few times with distilled water after loading.

2. Load the second and other well with 20 ml of VC-25 protein as described above. Do not pipette the pellet at the bottom of the microfuge tube. Rinse the syringe a few times with distilled water after loading.

5. Running the gel

1. Check that the buffer in the upper buffer chamber are full because leakage of the buffer may occur.

2. Place the lid on top of the lower buffer chamber. Make sure that the connection is correct, ie. black to black and red to red.

3. Attach the electrical leads to a suitable power pack with the proper polarity (black to black and red tored). Run the gel at a constant current of 30 mA.

4. Stop the electrophoresis when the tracker dye is ~ 1 cm above the end of the glass plates.

6. Removing and staining the gel.

1. Remove the gel from the buffer chamber

2. Loosen all four screws of the clamp assembly and remove the glass plate sandwich from it.

3. Push one of the spacers out to the side of the plates without removing it.

4. Gently twist the spacer so that the upper glass plate pulls away from the gel.

5. Cut the gel on one side (to orientate the gel).

6. Remove the gel by gently grasping two corners of the gel and place it in the container containing the Coomassie blue stain. Make sure that the gel is fully submerged in the staining solution.

7. Stain the gel for 1 hour, agitate it slowly on a shaker.

8. Destain the gel in a destaining solution a few times until protein bands are visualised.

9. Approximately determine the molecular weight of the visualised protein bands by comparing them with the molecular weight markers.

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Sorting of Proteins.

Protein Sorting

Protein synthesis is initiated on ribosomes in cytosol except for those which are synthesised on the ribosomes of plastids. Most proteins do not have sorting signals & remain permanent resident of cytosol. Many others have specific sorting signals that direct their transport to different organelles.

There are three fundamental ways of protein transport:

1. Active transport through pores.
2. Transmembrane transport by a membrane bound translocator.
3. Vesicular Transport.

All the three mechanisms are guided by sorting signals which are mainly of two types:

1. Signal Peptide : A continuous stretch of 15 – 16 amino acids on the polypeptide at either terminal, which may be cleaved post sorting. (fig.A). Signal peptide direct the protein from cytosol to various orgaelles
2. Signal Patch : A specific 3D arrangement of amino acids on protein surface, once it folds. This portion may not be a linear stretch of amino acids hence signal patch (fig.B) Signal patches identifyenzymes that glycosylates them. Once glycosylated, the sugar residue then direct such proteins from Golgi to other target organelles.

Organelle specific Sorting of Proteins

Peroxisomes

· All peroxisomal proteins are synthesised on cytosolic ribosomes and incorporated post translationally.

· Most proteins have signal sequences at C – terminal while a few have at N – terminal. Signal sequences are part of functional proteins, hence not cleaved.

· Signal sequences are specific 3 amino acid sequence at either terminal.

· Many proteins are imported in a folded state across the peroxisomal membrane. This translocation involves ATP hydrolysis.

· Proteins incorporated in peroxisomal membrane & peroxisomal matrix have different signal sequence.

Mitochondria and Chloroplast

· Plastid proteins that are encoded by nuclear genes & synthesised by cytosolic ribosomes are imported post-translationally, but in an unfolded state. Cytosolic chaperons maintain the unfolded state.

· Signal sequence lies at N-terminal & is always cleaved after transporting into matrix.

· Signal sequence for mitochondria have alternate specific positively charged amino acids at one terminal and hydrophobis at another.

· Signal sequence for chloroplast on the other hand is the protein itself, rich in serine, threonine and other small hydrophobic amino acid but poor in aspartate & glutamate.

· Translocation occurs at a site where outer and inner membranes are close together.

· Protein is first transported into matrix and then redirected to its destination. Hence two or more signal sequences (one directing transport from cytosol to plastid matrix and the rest direct it to its destination in plastid itself.)

Endoplasmic Reticulum (ER)

· Proteins are transported into ER either post-translationally or cotranslationally. Latter requires direct association of cytosolic ribosome to ER achieved by Signal Recognition Particle, SRP.

· While polypeptide is being synthesised, SRP recognises signal sequence, binds to it, then to ribosome and targets the whole to ER at a specific receptor.

· Binding of SRP to ribosome stalls translation temporarily. Once the whole assembly binds at ER, SRP unbinds from polypeptide signal & SRP receptor at ER. This allows resumption of polypeptide synthesyis by ribosome.

· The growing polypeptide is pushed into ER through a translocon channel and subsequently released into lumen of ER by cleaving signal sequence (always) by enzyme signal peptidase.

· In post translational transport, poypeptides that are destined to be permanent resident of ER, the signal sequence consists of specific 4 amino acids at C - terminal, wwhile those which are further directed into Golgi from ER have signal that comprises of 5-10 hydrophobic amino acids at N – terminal.

Details of co-translational sorting into ER.

Proteins that are sorted cotranslationally contain a signal sequence at N -terminal of the growing polypeptide. The main step in such transport is association of ribosome to ER membrane via a Signal Recognition Particle (SRP).

SRP is a G-protein consisting of 6 polpeptides & 7s RNA. Main steps of sorting are:

1. SRP binds to signal sequence of polypeptide and also ribosome, inhibits the translation.

2. It then targets the whole assembly of ribosome, mRNA, polypeptide, and itself to ER. There it binds at a SRP receptor on ER membrane. The ribosome also attaches itself to SRP receptor.

3. Shortly after SRP releases itself from assembly facilitated by GTP hydrolysis. The signal peptide is thus released into translocon. Translocon is made up of 3 membrane proteins – sec61, it is gated channel, 50’A in diameter

4. Release of SRP allows ribosome to resume translation. At this point ribosome completely blocks the translocon. The growing polypeptide is directly transferred into lumen of ER through translocon.

5. Signal Peptidase is bound to an internal site of translocon and serves the purpose of cleaving signal sequence.

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Swine Flu H1N1 - Pandemic History

There is pandemonium surrounding Swine Flu H1N here in India (where we love to ignore the deadly Malaria and TB). The number of deaths due to to Swine Flu is fraction of what every year malaria alone causes. But who cares about mosquito bites and the filth around.. Swine Flu is the 'In Thing'. Everyone from second class traveller in train to the daily Sea Link traveller is bothered about Swine Flu and has his/her personal opinion on it, given the Dailies are trying to educate generality of men through articles published. But we cant afford to know little. I am publishing this article about H1N1, its history and some properties etc... I am typing out this excerpt from textbook of immunology by Kuby. This is definitely more comprehensive than articles from newspaper and requires one to have some prior knowledge of viruses.

'The influenza virus infects the upper respiratory tract andmajor central airways in humans, horses, birds, pigs, andeven seals. In 1918–19, an influenza pandemic (worldwide epidemic) killed more than 20 million people, a toll surpassingthe number of casualties in World War I. Some areas,such as Alaska and the Pacific Islands, lost more than half oftheir population during that pandemic.'

PROPERTIES OF THE INFLUENZA VIRUS

Influenza viral particles, or virions, are roughly spherical or ovoid in shape, with an average diameter of 90–100 nm. Thevirions are surrounded by an outer envelope—a lipid bilayer acquired from the plasma membrane of the infected host cell during the process of budding. Inserted into the envelope are two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which form radiating projections that are visible in electron micrographs . The hemagglutinin projections, in the form of trimers, are responsible for the attachment of the virus to host cells. There are approximately1000 hemagglutinin projections per influenza virion. The hemagglutinin trimer binds to sialic acid groups on host-cellglycoproteins and glycolipids by way of a conserved amino acid sequence that forms a small groove in the hemagglutinin molecule.Neuraminidase, as its name indicates, cleavesN-acetylneuraminic (sialic) acid from nascent viral glycoproteins and host-cell membrane glycoproteins, an activity that presumably facilitates viral budding from the infected host cell.Within the envelope, an inner layer of matrix protein surrounds the nucleocapsid, which consists of eight dif-ferent strands of single-stranded RNA (ssRNA) associated with protein and RNA polymerase . Each RNA strand encodes one or more different influenza proteins.

Three basic types of influenza (A, B, and C), can be distinguished by differences in their nucleoprotein and matrix proteins.

Type A, which is the most common, is responsible for the major human pandemics. Antigenic variation in hemagglutinin and neuraminidase distinguishes subtypes of type A influenza virus. According to the nomenclature of the World Health Organization, each virus strain is defined by its animalhost of origin (specified, if other than human), geographical origin, strain number, year of isolation, and antigenic description

of HA and NA (Table 17-2). For example, A/Sw/Iowa/15/30 (H1N1) designates strain-A isolate 15 that arose in swine in Iowa in 1930 and has antigenic subtypes 1 of HA and NA. Notice that the H and N spikes are antigenically distinct in these two strains. There are 13 different hemagglutinins and 9 neuraminidases among the type A influenza viruses. The distinguishing feature of influenza virus is its variability. The virus can change its surface antigens so completely that the immune response to infection with the virus that caused a previous epidemic gives little or no protection against the virus causing a subsequent epidemic. The antigenic variation results primarily from changes in the hemagglutinin and neuraminidase spikes protruding from the viral

envelope. Two different mechanisms generate antigenic variation in HA and NA: antigenic drift and antigenic shift. Antigenic drift involves a series of spontaneous point mutations that occur gradually, resulting in minor changes in HA and NA. Antigenic shift results in the suddenemergence of a new subtype of influenza whose HA and possibly also NA are considerably different from that of the virus present in a preceding epidemic. The first time a human influenza virus was isolated was in 1934; this virus was given the subtype designation H0N1 (where H is hemagglutinin and N is neuraminidase). The H0N1 subtype persisted until 1947, when a major antigenicshift generated a new subtype, H1N1, which supplanted the previous subtype and became prevalent worldwide until1957, when H2N2 emerged. The H2N2 subtype prevailed for the next decade and was replaced in 1968 by H3N2.Antigenicshift in 1977 saw the re-emergence of H1N1. The most recentantigenic shift, in 1989, brought the re-emergence of H3N2, which remained dominant throughout the next several years. However, an H1N1 strain re-emerged in Texas in 1995, and current influenza vaccines contain both H3N2 and H1N1 strains. With each antigenic shift, hemagglutinin and neuraminidase undergo major sequence changes, resulting in major antigenic variations for which the immune systemlacks memory. Thus, each antigenic shift finds the populationimmunologically unprepared, resulting in major outbreaks of influenza, which sometimes reach pandemic proportions. Between pandemic-causing antigenic shifts, the influenzavirus undergoes antigenic drift, generating minor antigenic variations, which account for strain differences within a subtype.The immune response contributes to the emergence of these different influenza strains. As individuals infected with a given influenza strain mount an effective immune response, the strain is eliminated. However, the accumulation of point mutations sufficiently alters the antigenicity of some variants so that they are able to escape immune elimination . These variants become a new strain of influenza, causing another local epidemic cycle. The role ofantibody in such immunologic selection can be demonstratedin the laboratory by mixing an influenza strain with a monoclonal antibody specific for that strain and then culturing the virus in cells. The antibody neutralizes all unaltered viral particles and only those viral particles with mutations resulting in altered antigenicity escape neutralization and are able to continue the infection.Within a short time in culture, a new influenza strain can be shown to emerge. Antigenic shift is thought to occur through genetic reassortment between influenza virions from humans and from various animals, including horses, pigs, and ducks. The fact that influenza contains eight separate strands of ssRNA makes possible the reassortment of the RNA strands of human and animal virions within a single cell infected with both viruses. Evidence for in vivo genetic reassortment between influenza A viruses from humans and domestic pigs was obtained in 1971. After infecting a pig simultaneously with human Hong Kong influenza (H3N2) and with swine influenza (H1N1), investigators were able to recover virions expressing H3N1. In some cases, an apparent antigenic shift may represent the re-emergence of a previous strain that has remained hidden for several decades. In May of 1977, a strain of influenza, A/USSR/77 (H1N1), appeared that proved to be identical to a strain that had caused an epidemic 27 years earlier. The virus could have been preserved over the years in a frozen state or in an animal reservoir.

When such a re-emergence occurs, the HA and NA antigens expressed are not really new; however, they will be seen by the immune system of anyone not previously exposed to that strain (people under the age of twenty-seven in the 1977epidemic, for example) as if they were new because no memory cells specific for these antigenic subtypes will exist in the susceptible population. Thus, from an immunologicpoint of view, the re-emergence of an old influenza A strain. 

HOST RESPONSE TO INFLUENZA INFECTION

Humoral antibody specific for the HA molecule is produced during an influenza infection. This antibody confers protection against influenza, but its specificity is strain-specific and is readily bypassed by antigenic drift. Antigenic drift in the HA molecule results in amino acid substitutions in several antigenic domains at the molecule’s distal end. Two of these domains are on either side of the conserved sialic-acid–binding cleft, which is necessary for binding of virions to target cells. Serum antibodies specific for these two regions are important in blocking initial viral infectivity. These antibody titers peak within a few days of infection and then decrease over the next 6 months; the titers then plateau and remain fairly stable for the next several years. This antibody does not appear to be required for recovery from influenza, as patients with agammaglobulinemia recover from the disease. Instead, the serum antibody appears to play a significant role in resistance to reinfection by the same strain. When serum-antibody levels are high for a particular HA molecule, both mice and humans are resistant to infection by virions expressing that HA molecule. If mice are infected with influenza virus and antibody production is experimentally suppressed, the mice recover from the infection but can be reinfected with the same viral strain. In addition to humoral responses, CTLs can play a role in immune responses to influenza.

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Gene regulation - An Introduction

Gene expression can be controlled at any of several stages, which we divide broadly into transcription, processing, and translation:

• Transcription often is controlled at the stage of initiation. Transcription is not usually controlled at elongation, but may be controlled at termination to determine whether RNA polymerase is allowed to proceed past a terminator to the gene(s) beyond.

• In eukaryotic cells, processing of the RNA product may be regulated at the stages of modification, splicing, transport, or stability. In bacteria, an mRNA is in principle available for translation as soon as (or even while) it is being synthesized, and these stages of control are not available.
• Translation may be regulated, usually at the stages of initiation and termination (like transcription). Regulation of initiation is formally analogous to the regulation of transcription: the circuitry can be drawn in similar terms for regulating initiation of transcription on DNA or initiation of translation on RNA.

The basic concept for how transcription is controlled in bacteria was provided by the classic formulation of the model for control of gene expression by Jacob and Monod in 1961 (Jacob and Monod, 1961). They distinguished between two types of sequences in DNA: sequences that code for trans-acting products; and cis-acting sequences that function exclusively within the DNA. Gene activity is regulated by the specific interactions of the trans-acting products (usually proteins) with the cis-acting sequences (usually sites in DNA). In more formal terms:

• A gene is a sequence of DNA that codes for a diffusible product. This product may be protein (as in the case of the majority of genes) or may be RNA (as in the case of genes that code for tRNA and rRNA). The crucial feature is that the product diffuses away from its site of synthesis to act elsewhere. Any gene product that is free to diffuse to find its target is described as trans-acting.
• The description cis-acting applies to any sequence of DNA that is not converted into any other form, but that functions exclusively as a DNA sequence in situ, affecting only the DNA to which it is physically linked. (In some cases, a cis-acting sequence functions in an RNA rather than in a DNA molecule.

To help distinguish between the components of regulatory circuits and the genes that they regulate, we sometimes use the terms structural gene and regulator gene. A structural gene is simply any gene that codes for a protein (or RNA) product. Structural genes represent an enormous variety of protein structures and functions, including structural proteins, enzymes with catalytic activities, and regulatory proteins. A regulator gene simply describes a gene that codes for a protein (or an RNA) involved in regulating the expression of other genes.

..

Figure 10.1  
A regulator gene codes for a protein that acts at a target site on DNA.

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The simplest form of the regulatory model is illustrated in Figure above: a regulator gene codes for a protein that controls transcription by binding to particular site(s) on DNA. This interaction can regulate a target gene in either a positive manner (the interaction turns the gene on) or in a negative manner (the interaction turns the gene off). The sites on DNA are usually (but not exclusively) located just upstream of the target gene.

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