Name NIM/Class

: Nuris A Dwi Riansah : 100210103033/X

GENE EXPRESSION

Gene ekspresion is a chain complex process which involve a lot of living factor. Generally gene ekspresion can be started and arranged when before initiation transcription stage, howerver there is no one can decide when actually the regulation process started and because biology is a cycle and no one can find the start for sure. Controling gene expression is very important aspect for the living being , in the prokariotic or even eucariotic being, without this eficient control, cell will loss more of ebnergy. There are 2 system activiting gene expression that is constitutive and inductive. Constitutive means always expresed in every condition meanwhile inductive mean gene only expresed in the condition which allow the process happen. Constitutive gene is gene which reponsible for basic metabolism meanwhile the induction genes show the cellular eficiency. There are also some aspect which arrange the DNA to transcript or others, that called by name gene regulator or regulatory genes and the function of the regulatory genes is products, either RNA or proteins, interact with other sequences and affect their transcription or translation. In many cases, the products of regulatory genes are DNA binding proteins. Regulatory elements also affect the expression of sequences to which they are physically linked. Much of gene regulation takes place through the action of proteins produced by regulatory genes that recognize and bind to regulatory elements Based from the of the transcribed or regulated, the gene regulator devided into 2 types that is structural genes and regulatory genes. Structural genes are encoded proteins that are used in metabolism or biosynthesis or that play a structural role in the cell. Meanwhile Regulatory genes are, genes whose products, either RNA or proteins, interact with other sequences and affect their transcription or translation. In many cases, the products of regulatory genes are DNA binding proteins. In some condition there are some DNA sequence which arent transcribed but still have function in regulating nucleotide sequences. The proteins produced by regulatory genes that recognize and bind to regulatory elements.

DNA- Binding Proteins DNA- Binding proteins are proteins that are composed of DNA-binding domains and thus have a specific or general affinity for either single or double stranded DNA and can inuence their gene expression. DNA-binding proteins can be grouped into several distinct types on the basis of a characteristic structure, called a motif, found within the binding domain. There are 6 types of the DNA-binding proteins based om motive, that is Helix-turnhelix, Zinc-nger, teroid receptor, eucine-zipper ,Helix-loop-helix , and Homeodomain Major different of each DNA-binding proteins form Motiv type Helix-turn-helix Location Bacterial regulatory proteins; related motifs in eukaryotic proteins Zinc-nger Eukaryotic regulatory and other proteins teroid receptor Eukaryotic proteins Loop of amino acids with zinc at base Two perpendicular alpha helices with zinc surrounded by four cysteine residues eucine-zipper Eukaryotic transcription factors Helix of leucine residues and a basic arm; two leucine residues interdigitate Helix-loop-helix Eukaryotic proteins Two alpha helices separated by a loop of amino acids Homeodomain Eukaryotic regulatoryproteins Three alpha helices Major groove Major groove Major groove and DNA backbone Major groove and DNA backbone Major groove Characteristics Two alpha helices Binding Site in DNA Major groove

The form of the DNA-binding protein

Helix turn helix

zinc finger

Leucine zipper

Helix Loop Helix

Steroid receptor Homeodomain

(based from e-book benjamin A. Pierce Genetics A Conceptual Approach) Regulation in Bacterial Cells

In the procariot cell, there are known a group of genes which called operon. In eucariot doesnt know operon system because it arranged by a diferent promotor. In procariot gene expression only happen in the transcription level and in eucariot it happen from the transcription until after translation.gene controling in the living being is include positive and negative, in procariot the controling involve activity of the gene regulator. Positve controling in the an operon can be actived by production of gene expression regulator. Meanwhile if the operon genes in disactivated by gene regulator product is called negative control. There are 2 products of the gene rugalator, that is represor and activator both of it appear in the near of the gene operator. Gene operator will make a location which called operator location, but it doesnt give code for proteins. A gene may be regulated at a number of points along the pathway of information ow from genotype to phenotype . First, regulation may be through the alteration of gene structure. Modications to DNA or its packaging may inuence which sequences are available for transcription or the rate at which sequences are transcribed. DNA methylation and changes in chromatin are two processes that play a pivotal role in gene regulation. A second point at which a gene can be regulated is at the level of transcription. For the sake of cellular economy, it makes sense to limit protein production early in the transfer of information from DNA to protein, and transcription is an important point of gene regulation in both bacterial nd eukaryotic cells. A third potential point of gene regulation is mRNA processing. Eukaryotic mRNA is. fourth point for the control of gene expression is the regulation of RNA stability. The amount of protein produced depends not only on the amount of mRNA synthesized, but also on the rate at which the mRNA is degraded. fth point of gene regulation is at the level oftranslation, a complex process requiring a large number of enzymes, protein factors, and RNA molecule. All of these factors, as well as the availability of amino acids and sequences in mRNA, inuence the rate at which pro- teins are produced and therefore provide points at which gene expression may be controlled. . As i mentioned before, there are 2 types of operon function that is positive and negative. In an operon with negative control at the operator site, the regulatory protein is a repressor the binding of the regulator protein to the operator inhibits transcription. In a negative inducible operon, transcription and translation of the regulator gene produce an active repressor that readily binds to the operator. For transcription to take place, something

must happen to prevent the binding of the repressor at the operator site. This type of system is said to be inducible, because transcription is normally off (inhibited) and must be turned on (induced). Regulatory proteins frequently have two binding sites: one that binds to DNA and another that binds to a small molecule such as an inducer. Binding of the inducer alters the shape of the repressor, preventing it from binding to DNA. Proteins of this type, which change shape on binding to another molecule, are called allosteric proteins. As with inducible operons, repressible operons are economical: the enzymes are synthesized only as needed. Note that both the inducible and the repressible systems that we have considered are forms of negative control, in which the regulatory protein is a repressor. We will now consider positive control, in which a regulator protein stimulates transcription. In a positive inducible operon, transcription would normally be turned off because the regulator protein would be produced in an inactive form. Transcription would take place when an inducer became attached to the regulatory protein, rendering the regulator active. Logically, the inducer should be the precursor of the reaction controlled by the operon so that the necessary enzymes would be synthesized only when the substrate for their reaction was present. Positive operon could also be repressible; transcription would normally take place and would have to be repressed, In this case, the regulator protein would be produced in a form that readily binds to DNA and stimulates transcription. By combinig it togther, operons might exhibit positive or negative control and be either inducible or repressible. negative inducible, negative repressible, positive inducible, and positive repressible. To do so, learn the meanings of positive and negative control and inducible and repressible; then use logic to work out the details of whether the

regulatory protein is a repressor or an activator and whether it is produced in an active or inactive form. But in some condition, the negetive effect of this combination also sometime occur in the operon level which cause some disorder. The lac Operon of E. coli F . Jacob and J. Monoth has succesfull documenting gene controling system on the microorganism by observe the genetic determine from 3 emzymes causing the reconstruction of lactose in the Escherichia coli. If the bactery growth in the media contain glucose, the enzymes for lactose reconstruction ammount will be very few. And when the bactery growth in the media which contain lactose and without glucose the ammount of the lactose enzymes

will increase up to 1000 times. Jacob and Monod explain this system as example of gene arrangement which used to cells metabolism. Based from it Jacob and Monod identified 3 genes which determine structure and sntetic of the 3 enzymes. The enzymes named with gene structural, and given with sign Z (-galaktosidase), Y (-galaktosidase permeasa), and A (galaktosida transatilase). -Galactosidase is encoded by the lac Z gene, permease by the lacY gene, and transacetylase by the lacA gene When lactose is absent from the medium in which E. coli grows, only a few molecules of each enzyme are produced. In the lac operon, the lacZ, lacY, and lacA genes have. A common promoter (lac P) are transcribed together. reconstruction of lactose in the Escherichia coli (based from e-book benjamin A. Pierce Genetics A Conceptual Approach)

Upstream of the promoter is a regulator gene, lac I, which has its own promoter (PI). The lacI gene is transcribed into a short mRNA that is translated into a repressor. Each repressor consists of four identical polypeptides and has two binding sites; one site binds to allolactose and the other binds to DNA. In the absence of lactose (and, therefore, allolactose), the repressor binds to the lac operator site lac O (operon). In upstream of the structural genes is the lac promoter. RNA polymerase binds to the promoter and moves down the DNA molecule, transcribing the structural genes. When the

repressor is bound to the operator, the binding of RNA polymerase is blocked, and transcription is prevented. When lactose is present, some of it is converted into allolactose, which binds to the repressor and causes the repressor to be released from the DNA. In the presence of lactose, then, the repressor is inactivated, the binding of RNA polymerase is no longer blocked, the transcription of lac Z, lac Y, and lac A takes place, and the lac enzymes are produced. Several compounds related to allolactose also can bind to the lac repressor and induce transcription of thelac operon. One such inducer is isopropylthiogalactoside (IPTG). Although IPTG inactivates the repressor and allows the transcription of lac Z, lac Y, and lac A, IPTG is not metabolized by -galactosidase; for this reason it is often used in research to examine the effects of induction, independent of metabolism With different composition of the operon, the bactery which used to it can be mutated using partial diploid strain within Escherichia coli which contain 2 different DNA molecules: the full bacterial chromosome and an extra piece of DNA. By using different combinations of mutations on the bacterial and plasmid DNA, Jacob and Monod determined that parts of the lac operon were cis acting (able to control the expression of genes on the same piece of DNA only) or transacting (able to control the expression of genes on otherDNA molecules). Jacob and Monod from the experiment by using Escherichia coli found there are 3 types of the mutation. There are structural gene mutation, regulator gene mutation, and promoter mutation. Structural gene mutation is mutations which mapped to the lacZ or lacY structural genes and altered the amino acid sequence of the enzymes encoded by the genes. These mutations clearly affected the structure of the enzymes and not the regulation of their synthesis. Using the partial diploid,Jacob and Monod are able to establish that mutations at the lac Z and lac Y genes were independent and usually affected only the product of the gene in which they occurred. Regulator gene mutation is a mutations which affected the regulation of enzyme production. These mutations affected the production of both -galactosidase and permease, because genes for both enzymes are in the same operon and are regulated coordinately. This mutation can cause thelac enzymes to be produced all the time, whether lactose was present or not, and these mutations fell into two classes: regulator and operator. Promoter mutation is a mutation which affecting lactose metabolism, these mutations are designated lac P, and they interfere with the binding of RNA polymerase to the promoter.

Because this binding is essential for the transcription of the structural genes, E. coli strains with lac P mutations dont produce lac enzymes either in the presence or in the absence of lactose. Catabolite repression results from positive control in response to glucose. (This regulation in addition to the negative control brought about by the repressor binding at the operator site of the lac operon when lactose is absent.) Positive control is accomplished through the binding of a dimeric protein called the catabolite activator protein (CAP) to a site that is about 22 nucleotides long and is located within or slightly upstream of the promoter of the lac genes. Positive control is accomplished through the binding of a dimeric protein called the catabolite activator protein (CAP) to a site that is about 22 nucleotides long and is located within or slightly upstream of the promoter of the lac genes The trp Operon of E. coli Beside lac operon there are also trp operon, if lac operon is a system for lactose so trp operon is a system of th tryptophan. Tryptophan (trp) operon in E. coli, which controls the biosynthesis of the amino acid tryptophan, is an example of a repressible operon. The trp operon contains five structural genes (trp E, trp D, trp C, trp B, and trp A), which produce components of three enzymes (two of the enzymes consist of two polypeptide chains). These enzymes convert chorismate into tryptophan. Based from the observation by scientists, operon trp contain some promotoral area and a operator which placed in the next to the structural genes which give code to the enzymes. There is a promotor area which interacted with ARN polimerase, operator interacting with a represor molecule, if it bounded with tryptophan it will stop the structural genes transcription. In other word if there are tryptophan, some molecules amino acid will interacted with represor operon trp. This combination later will bound with DNA operator to stop the transcription. Which means if there are tryptophan within it so why need to make it again. Another unique aspect f trp operon is DNA area which located in between area operator and first structural gene (trp E). This area will keep transcripted and called leader area, transcription of the leader area will continue even represor molecules bound with the operator. The only one which can stop it is only interaction of the t-RNA mounted with tryptophan and the suite of short base in the end of the leader aera or called with atenuator. If t-RNA wasnt monted with tryptophan it wont stop until structural gene.

In trypttophan operon,th structural gene transcription arranged by 2 ways depend on the tryptophan within cell. If there arent tryptophan within cell, there wont tryptophan-represor substances to interacted with operator. Also there wont t-RNA mounted with tryptopan to interacted with atenuator area. Because the cell need to create tryptophan with enzymes which happen in the cell and interacted with represor molecules and t-RNA so the transcription will be stoped. The trp operon controls (based from e-book benjamin A. Pierce Genetics A Conceptual Approach)

Antisense DNA Regulation In DNA also found antisense, a small RNA molecules are complementary to particular sequences on mRNAs, which have function to control gene expression by binding to sequences on mRNA and inhibiting translation. The translation control which affected by antisense can be seen in the omp-F gene on E.coli, which allow the E.coli to adapt to external osmolarities. As i mentioned before, antisense also have function to inhibit translation. The translation which inhibited by reducing amount of of translation. For addition, antisense only found in the bacteria and bacteriophages.

Translation regulated by RNA antisense (based from e-book benjamin A. Pierce Genetics A Conceptual Approach)

Transcriptional Control in Bacteriophage Lambda Bacteriophage is a virus which can infect E.coli by inserting its DNA to the bacteria. Bacteriophage have a single DNA chromosome which consist of of 48,502 nucleotides sorounded by protein coat. The bacteriophages have 2 life cycles, there are lytic and lysogenic cycle. In lytic phase, the bacteriophages genes are transcribed and translated to produce protein and enzymes which syntetize 100-200 copies DNA. Meanwhile the lysogenic phase, the DNA not just transcribed, but it inserted to the other bacteria to encode and duplicated along with the bacteria chromosome. After it hatch from the bacteria once again it do the lytic again. In the lytic cycle, the genes that encode replication enzymes, phage proteins, and bacterial cell lysis are transcribed; but, in the lysogenic cycle, these genes are repressed.

(based from e-book benjamin A. Pierce Genetics A Conceptual Approach)

Eukaryotic Gene Regulation In eucaryiotic gene regulation system, there are many atribute which also can be found in the procaryotic organism. Some of it are : in both types of cells, DNA-binding proteins influence the ability of RNA polymerase to initiate transcription. But beside of the similiarity of both type of organism, there are also some atribute which make it completely different there are in procaryotic organism genes are organized into operon meanwhile in eucaryiot organism arent organized into operon and rarely transcribed togeteher into a single mRNA, in eucaryotic organism chromatine structure are affect gene expession, the function of the both represor and activator in eucaryotic more often use activator, and eucaryotic organism have greater diversity of mechanism genetics. In the nucleus of the eucaryotic organism, the histone proteins associate to form octamers, around which helical DNA tightly coils to create chromatin. The chromatin which formed from this process are usually used for a gene to be transcribed, transcription factors, activators, and RNA polimerase must bind to the DNA. From this part also make chromatin structure changes, and the DNA becomes more accessible to the transcriptional machinery. When genes become transcriptionally active , there are some changes which occur in the chromatin. One of it are the chromatine become more sensitive it si because the chromatin degradated by DNase I, an enzyme that digests DNA. And the ability of the Dnase I to able degradate chromatin proves DNA-histone association. The area which become more

sensitive in chromatine are called called DNase I hypersensitive sites, requently develop about 1000 nucleotides upstream of the start site of transcription, suggesting that the chromatin in these regions adopts a more open conguration during transcription. This relaxation of the chromatin structure may allow regulatory proteins access to binding sites on the DNA. One factor affecting chromatin structure is acetylation, the addition of acetyl groups (CH3CO) to histone proteins. Histones in the octamer core of the nucleosome have two domains: first is globular domain, globular domain is a domain that associates with other histones and the DNA. Second is a positively charged tail domain that probably interacts with the negatively charged phosphates on the backbone of DNA. Acetyl groups are added to histone proteins by acteyltransferase enzyme by neutralizing the positive charges on the

histone tails and allowing the DNA to separate from the histones. Beside acteyltransferase enzym there are also other enzymes called deacetylases strip acetyl groups from histones and restore chromatin repression. Some transcription factors and other regulatory proteins are known to alter chromatin structure without acetylating histone proteins. These chromatinremodeling complexes bind directly to particular sites on DNA and reposition the nucleosomes, allowing transcription factors to bind to promoters and initiate transcription. Another change in chromatin structure associated with transcription is the methylation of cytosine bases, with the repression of transcription in vertebrates and plants, whereas transcriptionally active DNA is usually unmethylated in these organisms Transcriptional Control in Eukaryotic Cells Same with procaryotic organism, in eucaryotic organism Transcription is an important level of control in eukaryotic cells, and this control requires a number of different types of proteins and regulatory elements. Transcriptional activator proteins are required to bring about normal levels of transcription. These proteins bind to a regulatory promoter, which is located upstream of the core promoter, and to enhancers, which may be located some distance from the gene. Transcriptional activator proteins stimulate transcription by facilitating the assembly or action of the basal transcription apparatus at the core promoter; meanwhile the activators gene may interact directly with the basal transcription apparatus or indirectly by using coactivator. There are 2 main function of transcriptional activator first is to binding DNA at specific bas squence. Second is to interact with other component of of transciptional apparatus and influence the transcription rate. GAL4 is a transcription activator protein that regulates the transcription of several yeast genes in galactose metabolism. Its contain some zinc finger and binding with sequence called UASg. When bound to UASG, GAL4 stimulates the transcription of yeast genes needed for metabolizing galactose. GAL4 and a number of other transcriptional activator proteins contain multiple amino acids with negative charges that form an acidic activation domain, and it can stimulate transcription by enhancing the ability one of the general transcription factors, to join the basal transcription apparatus. Transcription is activated by GAL4 in response to galactose. (based from e-book benjamin A. Pierce Genetics A Conceptual Approach)

In eucaryotic organism are also have known represor to inhibiting the transcription. The work of the represor repressors is to bind with sequences in the regulatory promoter or to distant sequences called silencers, which, like enhancers, are position and orientation

independent. But it have some different charachteristic with represor which appear in procaryotic organism especially when facing with RNA polymerase, it didnt directly block RNA polymerase. Repressors may compete with activatorsfor DNA binding sites: to stimulate transcription. A also repressor may bind to sites near an activator site and prevent the activator from contacting the basal transcription apparatus. There are also some posibility action of represor is direct interference with the assembly of the basal transcription apparatus. Some activator proteins bind to enhancers, which are regulatory elements that are distant from thegene whose transcription they stimulate. By binding activator proteins and cause the DNA between the enhancer and the promoter to loop out, bringing the promoter and enhancer close to one another, so that the transcriptional activator proteins are able to directly interact with the basal transcription apparatus at the core promoter Insulators are DNA sequences that block the action of enhancers.

Eventough eucaryotic organism genes doesnt contain operon, it still can be activated by same manners with procaryotic organism gene. It can be happened because they have regulatory sequences in common in their promoters or enhancers, such common DNA regulatory sequences are called response elements; they typically contain short consensus sequences. A single eukaryotic gene may be regulated by several different response elements.

REFRENCE Pai, C. Anna, 1985, THE BASICS OF GENETIC translated by Muchidin Apandi, Jakarta, Erlangga Pierce, A. Benjamin, Genetics A Conceptual Approach E-book