CHBE ENGINEERING APPLICATIONS IN BIOTECHNOLOGY

2011-12 fall semester Prof.Dr. Zmrt Begm gel

Chapter 1 Introduction (Power Point presentation-Week1) Chapter 2 Basic Principles of Industrial Biotechnology Biological systems employed in industrial biotechnology E. coli, S. cerevisiae, filamentous fungi., eukaryotic cell cultures DNA, RNA and protein synthesis Structure of DNA and RNA & DNA Replication Gene organization Transcription, translation Regulation of gene expression Protein targeting Chapter 3 Industrial Strain/Process Improvement Techniques Classical techniques Molecular techniques Gene cloning methods Vectors PCR Site-directed mutagenesis Directed evolution DNA sequencing Chapter 4 Applications of Industrial and Agricultural Biotechnology Microbial systems Eukaryotic systems GMO detection Regulating and patenting food biotechnology

References: 1. Glick, BR and Pasternak, JJ Molecular Biotechnology: Principles and Applications of Recombinant DNA., ASM Press, USA. (most recent edition) 2. Lecture Notes (will be provided by e-mail) 3. Books on Biochemistry, Molecular Biology, Biotechnology...

CHAPTER 2 Basic Principles of Industrial Biotechnology

2.1 Biological systems In bioprocesses, cells, tissue cultures, or even enzymes are employed in reaction(s) that lead to product formation. Enzymes Enzymes are proteins having the ability of catalyzing biochemical reactions. They are, thus, biological catalysts. A wide range of enzymes are produced by microorganisms, as well as plants and higher eukaryotes. These enzymes can be purified, or partially purified for use in bioprocesses individually or in mixtures of more than one enzyme. One example is the production of HFCS (high fructose corn syrup) from corn starch, where enzymes like -amylase, glucoamylase and glucose isomerase are used in pure or partially purified forms. An enzyme may also be the product of a bioprocess, e.g. production of glucoamylase by Aspergillus niger. Purified enzymes may be used in free or in an immobilized form. Immobilization is achieved by fixing enzymes on a solid support. The type of solid support and the way by which enzymes are attached onto the solid support is variable. Enzymes may be linked to the solid support by ionic interactions or by covalent linkages. Alternatively, they may be physically entrapped into porous particles or a matrix support. The method by which enzymes are immobilized depends on the particular reaction, as well as the stability and kinetics of the enzyme. Enzymes have catalytic power due to the presence of an active site in their 3-dimensional structure. The substrate of the enzyme binds the active site where electron transfer reactions take place leading to the conversion of substrate into product. When the product is formed, it leaves the enzyme and the same enzyme molecule continues to catalyze further reactions until the enzyme looses its activity by inhibition or loss of stability. Genetic and protein engineering techniques may be used for the aim of enhancing the stability of enzymes, or for increasing their catalytic power. One of the most important approach is

site-directed mutagenesis (SDM). Among the SDM techniques, specifically directed evolution methods are most widely employed. A restriction enzyme. In this representation the secondary (alpha-helices, beta-strands...etc.) and tertiary structure is shown. The substrate (in this case DNA, is also shown) Trypsin (a protease) This is only a different kind of representration whereby the amino acid residues are also visible. Bacteria Bacteria are prokaryotic organisms. Both Gram(+) and Gram(-) bacteria are employed in bioprocesses. One example is the production of human insulin by Escherichia coli, and another one is the production of thermostable -amylase by Bacillus stearothermophilus. The former is a recombinant organism carrying the human insulin gene, while the latter is a natural isolate which has underwent strain improvement to enhance -amylase production. Bacillus species are also used for the production of proteases used in detergents. Another group of bacteria, of importance to biotechnology, are the lactic acid bacteria, which are widely used in food fermentations, such as in yoghurt and pickled vegetable and meat production. Acetic acid bacteria are also employed, especially for the production of vinegar (Acetobacter spp.). The Prokaryotic Cell Yeast Yeasts are unicellular eukaryotes that are part of the fungal kingdom. Yeast cells are slightly bigger than bacteria (bacteria: 0.5-3 m, yeasts: 5-10 m). Reproduction of these cells is most commonly by budding, but also by fission or other types of sporulation. During fermentation reproduction is predominantly in the form of budding. S. cerevisiae and its close relatives are used as brewers and bakers yeasts. S. cerevisiae is a facultative anaerobe, thus an aerobic organism which can also grow under anaerobic conditions. Fermentation is a process where growth takes place under anaerobic conditions.

For example, wine and beer production are anaerobic processes, while the production of bakers yeast is a aerobic process. The Yeast Cell

The budding yeast. The major form of yeast replication Filamentous fungi (molds) These are eukaryotes that belong to the fungal kingdom, like the yeasts. However these organisms have a highly branched, filamentous (mycelial) structure. Individual filaments are called hyphae (pl. hypha). Hypha consist of many cells attached to each other to form long chains of cells separated by a barrier, called septum. Thus cells within the mycelium are in a way compartmentalized. Nevertheless, in certain types of filamentous fungi septa is not encountered.

Reproduction takes place both by asexual and sexual spore formation. The asexual spores are called conidia. Different molds produce different types of spores. Examination of spore morphology is one method by which molds are identified and classified; e.g. the perfect forms of Aspergillus species are among the ascomycetes. Spores are formed on an ascocarp, bearing ascospores. Mushroom is the fruiting body of a certain class of filamentous fungi, called the basidiomycetes. The mushroom industry is one of the important sectors of biotechnology. The major edible mushroom is Agaricus bisporus. Certain mushrooms are produced for the production of pharmaceuticals. A large fraction of the bioprocess industry employs molds, due to their ability of secreting large amounts biochemicals and enzymes; e.g. Penicillin production by Penicillium chrysogenum. Utilization of Penicllium species in cheese production. Production of enzymes, such as cellulases, ligninases, -amylase and glucoamylase, proteases, invertase, glucose isomerase by Aspergillus and other molds.

The fungal mycelium (hyphae) viewed under the optical microscope.

Photosynthetic organisms Other than plants cyanobacteria (prokaryotic) and algae (eukaryotic) are also photosynthetic organisms. During photosynthesis sunlight is converted into complex organic compounds, i.e. light energy is converted into electrical energy by an electron flow that causes the separation of negatively and positively charged molecules. A detailed understanding of this process may help to recreate this important biological function. One application could be the production of solar-powered electrolytic battery. Some biotechnological applications of algae involve the production of agar, alginic acid, waste-water treatment (spent algae can be used in the production of SCP (single cell protein) used as animal feed). Sources of microorganisms Microbial cultures can be ordered from culture collections. Most famous ATCC (American Type Culture Collection) NRRL (Northern Utilization Research and Development Division) CMI (Commonwealth mycological Institute) Turkey TBTAK (There is a rich collection of fungi, and especially molds) Plant cell and tissue culture The in vitro culture of cells, tissue and organs of plants is one of the growth areas of biotechnology. Major aim is to generate improved crops and ornamental plants. Plant cells are also used for the production of pharmaceuticals, hormones and other products, like flavoring compounds. Genetic techniques are used for the generation of genetic variants, as well as the selection, maintenance and propagation of desired variants. Plant growth and development seed germination

seedling emerges stem and root with apical growth region and branches (these originate as buds and form leaves and flowers) At the apical growth region there are meristematic cells, which are undifferentiated cells and sources of all cell types in the primary tissues. Plant tissue culture In order to make a tissue culture, small pieces of living tissue (called: explants) are isolated and grown aseptically on a nutrient medium. It is best to choose explant rich in undifferentiated cells, e.g. meristem or cortex. Usual explants are buds, root tips, germinating seeds...etc. Such undifferentiated cells are capable of rapid proliferation on nutrient media, and result in the formation of an undifferentiated mass of cells, known as callus. Thus; explant + culture media callus undiffrentiated mass of cells can be propagated indefinately by subdivision

The Plant Cell Plant cell culture When callus is transferred to a liquid medium and agitated, cell mass breaks up, and forms a suspension of isolated cells capable of division. Single cells can likewise be plated out on solid media to give a callus. Major problems, about plant cell cultures, are aggregation and genetic instability, which causes heterogeneity. One of the solutions to the problem is the use of tobacco and soybean cell lines, which are relatively homogeneous. Furthermore, cells can be readily cultivated batchwise and continuously, and can be immobilized. Another problem about the use of plant cell cultures is contamination. Since these cells grow more slowly than microbial cells, growth is easily overtaken by microorganisms. Using aseptic conditions is the solution to the problem, however, this is often not so easy for largescale operations. Providing complete asepsis in a factory is not economic and requires skilled labor. Plant cells are used in the production of fine chemicals, insecticides, perfumery, food flavorings, and colorants. 2.2 DNA, RNA and Protein Synthesis Structure of DNA and RNA and DNA Replication Figures are shown in the class room. + DNA Replication Animation from Russell Note these concepts: DNA is double-stranded, strands run in anti-parallel manner. In replication, each strand is used as a template; one is the leading strand the other is the lagging strand. RNA primer is required for the initiation of replication, okazaki fragments are synthesized during the replication of the lagging strand. Replication is semi-conservative (one of the strands of the newly synthesized DNA originate from the parent DNA molecule).

Helicase unwinds the DNA helix. DNA-Polymerase (DNA-Pol) synthesizes the DNA and performs proof-reading and error-correction. There are more than one DNA-Pol that perform these functions. Gene Organization and Transcription In a region of double-stranded DNA, the two strands run in opposite directions, as shown below; 5 3 3 5

Only one of these strands contains the genetic information in the correct reading frame (a reading frame indicates the codons of a gene). This strand is called the sense strand, and its complementary strand is called the anti-sense strand. An anti-sense strand is a noncoding strand, however, it is important in that it acts as the template of transcription. RNA polymerase, uses the anti-sense strand as the template to synthesize an mRNA copy of the sense strand. Thus, it is the sense strand that is copied for protein synthesis. If a gene is sued in protein synthesis, it is said to be expressed. Gene expression: transcription and translation for protein synthesis. The below scheme represents the organization of the elements on a gene required for gene expression: 5 TC TL p tL tC 3

p; promoter, where RNA polymerase binds for the initiation of transcription. Promoters are specific DNA sequences that are recognized by RNA polymerase. These are the essential control elements of gene expression. In the absence of a functional promoter, a gene can not be expressed. The sequence of the promoter determines the frequency by which transcription takes place, because this effects the affinity of RNA polymerase to the promoter. Strong promoters yield high level of expression, and vise versa. Nevertheless, the promoter is not the only control element of gene expression. Control (regulatory) sequences may exist upstream or downstream of a gene. These elements may have functions such as turning the gene expression on or off, induction, repressionetc.

In general, promoters contain some consensus sequences (common sequences) such as the socalled TATA box, CAAT box, etc. , but the overall sequence of the binding region is variable. TC and tC; transcription initiation and termination sites, respectively. These are sites where transcription is initiated and terminated by RNA polymerase. Initiation of transcription is determined by the location of the promoter. Transcription termination is recognized by specific DNA sequences that allow transcription termination to take place. TL and tL; translation initiation and termination sites, respectively. Translation is initiated by a methionine (M or Met) codon (ATG) and is terminated by a stop codon (UAA, UGA or UAG). Each codon codes for a specific amino acid. Exercise. If a protein consists of e.g. 300 amino acids what are the number of codons? What is the number of bases of the coding sequence? It can be easily seen that, after transcription, the resulting mRNA transcript does not contain the promoter sequence anymore and is flanked by the transcription initiation and termination sites. These sites are slightly upstream and downstream of the translation initiation and termination sites (the coding region). Therefore, an mRNA transcript contains some sequences that are not translated. These extra sequences probably give some space for the enzymes and various molecules to work on the mRNA during the translation process. The 5 untranslated region is called the leader and the 3 untranslated region is called the trailer. Introns and mRNA modifications It is important to note that in prokaryotic genes the coding region is uninterrupted (codons follow one after the other) whereas in eukaryotic genes, the coding region is generally interrupted by non-coding sequences that are called introns; codons are interrupted by noncoding bases. The remaining coding sequences are called exons. Such genes are named as split genes. TL E1 I1 E2 I2 E3 tL 5

In the above figure, a split gene, consisting of three exons and two introns, is shown. Since the introns would normally create a problem during translation, the introns are removed, by a process called intron splicing, prior to translation. Thus, introns are present on the initially synthesized mRNA (pre-mRNA) just after transcription but, they are removed before translation to form the mature mRNA which contains the correct reading frame (codons are arranged such that the resulting protein has the desired amino acid sequence). In fact, this is not the only modification to eukaryotic mRNAs. Further modifications take place at the 5 and 3 ends. The 5 end is methylated and a poly-A tail, consisting of several A nucleotides (20 to several hundred), is added to the 3 end. The poly-A tail is added by the enzyme poly-A polymerase. (Note that the poly-A tail is used in genetic engineering to synthesize a DNA copy (cDNA) of a eukaryotic mRNA. This is generally the first step of a cloning procedure) Below is a scheme representing the mature mRNA of the split gene shown above:
5 cap

E1

E2

E3

AAAAAAAAAAAA3 3

In the formation of 5 cap, a GTP is added to the 5 ppp (tri-phosphate) resulting in the formation of an unusual 5-5 triphosphate linkage. The function of 5 cap and poly-A tail is not clear, however, there is evidence that they may be involved in the stimulation of translation. The prokaryotic mRNAs, on the other hand, have the following ends: 5 ppp The operon concept Another difference between the prokaryotic and eukaryotic mRNAs is that the prokaryotic mRNAs may be polycistronic (carrying the transcript of more than one gene), whereas the mRNAs of eukaryotes are always monocistronic (containing the transcript of a single gene). This is due to the lack of operons in eukaryotes. The operons in prokaryotes are regions where a number genes, with related function, are found together one after the other. A well-known OH 3

example is the lac operon which contains genes coding for enzymes involved in lactose utilization. The transcription process RNA polymerase binds the promoter of a gene and initiates transcription from the transcription initiation site. Elongation phase continues until transcription termination signals are encountered. In prokaryotes there are two major types of transcription termination signals. In both types, elongation ends once a terminator sequence is reached, however, in one type the release of RNA polymerase requires a protein factor, called rho, whereas in the other type release is rho-independent. In eukaryotes transcription termination mechanism is unclear, however, it is known that the initially synthesized mRNA has a long trailer sequence which is later cleaved by poly-A polymerase from a specific sequence that acts as the polyadenylation signal. As previously mentioned, this is followed by the incorporation of several As at the 3 end of mRNAs. In order to perform the transcription process RNA polymerase has the following requirements: 1. a template (double-stranded (ds-) or single-stranded (ss-) DNA) 2. Activated precursors (ATP,CTP,GTP,UTP or briefly NTPs) 3. Divalent metal ion (Mg2+ or Mn2+) The polymerization reaction proceeds in the following manner: (RNA)n + NTP RNA polymerase (RNA)n+1 + PPi

Polymerization occurs from 5 to 3 while running on the template from 3 to 5. Exercise: Find the answers to the below questions from your book (remember that students are responsible not only from lecture notes but also from all exercise questions and reading assignments) 1. Compare RNA polymerases of prokaryotes and eukaryotes. 2. Find out the details of the process of intron splicing. 3. Find out about the transcription of ribosomal RNA (rRNA) genes.

The Genetic Code and Translation In all the organisms that exist in the world, the same genetic language is used. This is a 4letter (A,C,G, T) alphabet yielding 3-letter words (codons). Each codon specifies one of the 20 amino acids. Yet, this simple alphabet is sufficient to create the enormous variety of organisms with much complexity. This is because of the fact that most proteins consist of 100s of amino acids. Thus, the number of proteins that can be made, is essentially unlimited. Exercise: From your book find the table showing the genetic code and work out the number of codons for each amino acid. What are the amino acids with the lowest and highest number of codons? Since more than one codons may specify the same amino acid, the code is said to be degenerate (redundant). Components of translation The components of translation are mRNA, ribosomes and the tRNAs. Ribosomes consist of ribosomal proteins (r-proteins) and ribosomal RNAs (rRNA). Almost 85% of the total cellular RNA consist og rRNAs because of their extensive use in the construction of ribosomes and also because of the requirement of large amounts of ribosomes for the translation of mRNAs. These requirements are maximum during the exponentail phase of cellular growth where gene expression is most extensive. In prokaryotes there are three types of rRNAs (23S, 16S, 5S) and in eukaryotes there are four types (28S, 18S, 5.8S, 5S). The ribosomes Ribosomes consist of a large and a small subunit. The prokaryotic ribosome (70S) consists of a 50S large subunit and a 30S small subunit. The large subunit consists of the 23 rRNA, 5S rRNA and 34 r-proteins, while the small subunit consists of the 16S rRNA and 21 rproteins. tRNAs tRNAs act as adaptor molecules to bring together the codons on the mRNA and the corresponding amino acids. Each tRNA has one side that fits the codon (called the anticodon) and one side that fits the amino acid. The attachment of amino acids to tRNAs are catalyzed by amino acyl-tRNA synthetases. There is at least one specific synthetase for each of the 20 amino acids. A tRNA having an attached amino acid is called a charged tRNA and without an attached amino acid it is called uncharged tRNA. The figure below shows the position of the amino acid and the anticodon on a tRNA

Amino

acid 3

anticodon region The anticodon consists of three bases and is complementary to the codon on the mRNA. 3 A 5 5 G

e.g.

anticodon of the tRNA 3 mRNA

U G C

The wobble hypothesis At least 61 tRNAs should exist but this is not the case. The number is much less. It has been shown that a single tRNA species can recognize several different codons. This is explained by the wobble hypothesis: 1. tRNAs may contain unusual bases, such as inosine (I). Inosine is a modified form of adenine and can form hydrogen (H-) bonds with A, U or C. 2. The 5 end of the anticodon does not have strict constraints on base-pairing, allowing Hbond formation with any of several bases located at the 3 end of the codon. The below combinations are possible: 5 end of anticodon G C A U I Exercise: 1. What is the minimum number of tRNAs required for the amino acid Serine? 3 end of codon U or C G U A or G A, U, or C

2. Find out the structure of inosine and compare it with that of adenine.

Codon bias As we have seen, most amino acids are coded by more than one codon, however, it appears that not all codons are used at an equal frequency but, some occur more often in gene sequences than the others. This is called codon bias (or codon preferance) and is gene and organism-specific. Question: Highly expressed genes contain those codons that are more biased (more frequently used). Why? Explain. The translation process Translation takes place in three stages: 1. Initiation (AUG): In prokaryotes although the first codon is that of methionine, a modified version of methionine (formyl- methionine or fMet) is inserted into the protein sequence. In eukaryotes this process does not take place and methionine is used instead. In some prokaryotic genes and some of the genes of the eukaryotic mitochondrial genome, instead of AUG, CUG or GUG may act as the initiating codon, but this is fairly exceptional. In eukaryotes, the ribosome is believed to bind the 5 cap of the mRNA and slide down until it reaches the first AUG. In prokaryotes, ribosome binding is different. There is a ribosomebinding site (RBS) on the mRNA where the ribosomal small subunit binds to initiate translation. The RBS is a sequence that is complementary to part of the 16S rRNA which is a component of the small ribosomal subunit. Such a mechanism allows internal initiation of translation to take place, which is essential for polycistronic mRNAs. Exercise: Go back to your notes about operons in prokaryotes and try to clearly understand the last sentence. The initiation phase is fairly complex and involves the action of many proteins called initiation factors (IF 1, 2, 3). This is an energy-requiring process where one mole of GTP is consumed. In fact more GTPs are consumed during the elongation step. 2. Elongation: Codons are read one next to the other from 5 to 3 on the mRNA. The large ribosomal subunit comes into play once the small subunit stops at the initiating AUG

together with the corresponding tRNA. The large subunit contains two sites for the attachment of tRNAs. These are called the P site and the A site. In the incoming tRNA enters the A site and the previous tRNA is on the P site. At this stage the amino acid(s) are linked by a peptide bond by peptidyl transferase, and one of the tRNAs becomes uncharged and leaves the ribosome. At this stage the ribosome slides one codon down the mRNA and the A site becomes the P site. The new A site becomes now available for the next tRNAetc. 3. Termination: once a stop codon is encountered, the ribosomal subunits disintegrate and leave the mRNA. For the figure showing entire translation process please refer to your hand-out or your book. The polysome The same mRNA can be translated by several ribosomes at the same time, one coming next to the other. This is called the polyribosome, or simply polysome. Furthermore, in prokaryotes transcription and translation may be coupled, whereas in eukaryotes these processes take place separately because of the presence of the nuclear membrane. 2.3 Regulation of Gene Expression What is gene regulation and why is it required? So far we have seen that transcription is initiated by RNA polymerase binding to the promoter and is followed by elongation and finally termination. Likewise, the translation process also requires necessary factors for initiation, elongation and termination. Following translation, proteins generally undergo further processes like post-translational processing and protein targeting. Regulation of gene expression may take place at any step of protein synthesis or the post-translational stages. It is a well organized process that requires particular signals to be inplace. For example, a gene can never be expressed if RNA polymerase fails to bind the promoter. A certain signal may prevent/allow the binding of RNA-Pol to the promoter, and thereby regulate gene expression. One example for this case is the lac operon which encodes the proteins required for lactose utilization. In this case, the signal is lactose, i.e. only in the presence of lactose there is efficient RNA-Pol binding to the promoter.

Some parts of the genome are devoted to gene regulation. This means that there are regions for the binding of regulatory proteins. In the case of the lac operon, there is a region just next to the promoter (upstream of the gene= at the 5 end of the sense strand). Regulatory regions may be upstream, downstream of a gene, inside the coding region of a gene, or, in some cases very far away from the gene. Gene expression is an energy-requiring process. Therefore, gene expression must be strictly controlled. It means, a gene should be expressed only if the encoded protein is essential for the cell under the existing conditions. Otherwise, the production of that particular protein will be a waste of energy, which is a competitive disadvantage in terms of the efficient utilization of the available energy resources. If a gene is expressed regardless of the available nutrients and environmental factors, this is called a constitutive gene. Only few genes are constitutively expressed. These are often genes that encode molecules which directly or indirectly have vital functions for the cells. In complex organisms where different tissues exist in different organs, regulation of gene expression is the explanation of the existing variation, despite of the presence of the same genetic information in each cell (i.e. all the 46 chromosomes of a human being are present in each cell but different genes are expressed in different cells). Factors influencing gene regulation In the case of microorganisms, gene regulation is mainly influenced by the following factors: 1. Cellular and environmental factors 2. Factors related to the gene, its transcript and translation product Cellular and environmental factors - the growth phase of a microorganism; i.e. lag, exponential (log), late exponential early stationary, stationary, death phase. During the different phases of a microorganism there will be different needs. For example, lag phase is an adaptation period with little growth. Thus, energy-producing metabolic pathways, cell division hence DNA replication..etc. are not very active yet. During the log phase there is rapid growth, energy-producing metabolic pathways are very active, hence there is cell division, DNA replication and gene expression is at its maximum. During the late log phase perhaps some genes are repressed (their expression is

stopped or minimized) but may be others are induced perhaps for the synthesis of storage compounds, for sporulation..etc.. The stationary phase is by no means a period where no gene expression takes place, but rather, secondary metabolites are produced (e.g. antibiotics,aflatoxins...etc.). The death phase is where obviously the metabolic functions are minimized or non-existant. -environmental factors; presence or absence of inducers, repressors, inhibitors..etc. Microorganisms survive on the nutrients that are available in the environment. There are a great range of carbon sources that can be utilized by a microorganism, including starch, cellulose, sucrose, lactose, glucose ..etc., however which of these compounds are present and at what concentration, depends on the particular environmental conditions. The same is also valid for the nitrogen sources and other nutrients. So, the microorganism should know which nutrients are available and should adjust its metabolism accordingly. This indicates that the genes of compounds essential for the particular environmental conditions should be expressed, and those that are unnecessary should not be expressed. This is essential for the optimal utilization of energy. Gene expression may also be influenced by the presence of toxins or inhibitors..etc. A carbon source may be available but its utilization may be negatively effected by the presence of an inhibitor that specifically inhibits a certain enzyme activity..etc. The Lac Operon The lac operon is a gene cluster consisting of three genes each coding one of the enzymes required for lactose utilization. As you remember, an operon is a cluster of genes connected to the same promoter and, hence, their transcription takes place at the same time. Their translation, however, takes place separately. In the case of the lac operon the three genes code for -galactosidase (z), galactoside permease (y) and thiogalactoside transacetylase (a). Since they are connected to the same promoter their transcription is coordinately regulated. The mechanism is as follows: In the absence of lactose, a repressor protein (coded by the I gene) is synthesized and binds to the operator (the control element) (o) which is downstream of the promoter (p) but also partly overlaps with the promoter. Thus, if the repressor binds the operator this prevents RNA polymerase binding to the promoter and transcription does not take place. However, when there is lactose in the medium, it inactivates the repressor which becomes unable to bind the

operator. Thus, RNA polymerase can bind the promoter and initiates transcription. Since the enzymes coded by the lac operon are required only in the presence of lactose, the logic behind this simple and clever mechanism is to prevent the expression of the lac operon in the absence of lactose. The mechanism is called induction.
promoter operator repressor gene i p o structural genes

no Tc Tc

X
repressor lactose

inactive repressor Another question is what happens if there is both lactose and glucose present in the

medium?. Certainly, since glucose is a monosaccharide and lactose is a disaccharide, the cells will prefer to consume glucose first and then lactose. In this case induction of the lac operon does not take place until glucose concentration falls to a critical level. Such a control mechanism is called catabolite repression (or glucose repression). The presence of glucose, or any other easily metabolized compound, does not only repress the lac operon. Rather, this is a more global control mechanism that effects many genes. Basically, the mechanism is as follows: A regulatory protein called CAP (catabolite activator protein) forms a complex with cAMP (cyclic AMP). In the presence of high levels of CAP-cAMP complex, genes that are under the control of catabolite repression are induced. High levels of glucose prevent CAP synthesis and thus the level of CAP-cAMP decreases. In the lac operon, CAP-cAMP binds to a site immediately upstream of the promoter and this extremely enhances RNA polymerase binding to the promoter. When the three cases are compared in terms of transcription efficiency: No lactose 10 Lactose (+) CAP-cAMP (-) 58 Lactose (+) CAP-cAMP (+) 3000

Thus, very little expression is still present even in the absence of lactose which is necessary to initiate the induction process. Also note that the inducer is not lactose but allolactose which is

synthesized from lactose by -galactosidase. In genetic engineering studies the lac operon is often used as a marker in transformation studies, as we shall see. In such studies, instead of allolactose, another more effective chemical, IPTG (isopropylthiogalactoside) is used as the inducer. Exercise: Read the regulation of trp operon expression from your book and compare the mechanism with that of the lac operon. Then, answer the following questions: -is this an induction mechanism? -what is the role of the leader sequence? -what is the role of RNA secondary structure formation? -why is the lac operon induced in the presence of lactose but trp operon is repressed in the presence of tryptophan? Factors related to the gene, its transcript and translation product -Gene copy number: The number of copies of a particular gene per chromosome can effect gene expression because the higher the number of copies of a gene the more is the amount of transcript produced per unit time. This is a typical approach in genetic engineering where gene copy number is deliberatly increased in some cases to enhance expression. However, studies also show that there is a limit to the copy number of a gene. Above a certain gene dosage, excessive utilization of the available expression machinery for the expression of a single gene, upsets the entire system resulting in the decrease of cell growth and productivity. -Promoter strength and promoter specificity: This is a very important concept in terms of genetic engineering studies. a. Promoter strength indicates that the sequence of the promoter determines the affinity of RNA-polymerase binding to a promoter. The higher the affinity the higher will be the frequency by which RNA-polymerase initiates transcription from that promoter. Remember that there exist consensus sequences for prokaryotic and eukaryotic promoters (-35 and 10 elements, TATA box..etc. Generally, a promoter with a consensus sequence is likely to be an average promoter. Deviations from the consensus sequence may either decrease or increase promoter strength. In genetic engineering studies it is mostly useful to work with strong promoters to enhance transcription, hence expression efficiency. However, others may also be useful for particular applications.

b. Promoter specificity: In prokaryotes, RNA-polymerase requires the so-called sigma factor for the initiation of transcription. The sigma factor of different bacteria appear to have differences and, therefore, the promoter of e.g. E. coli may not be functional in e.g. Bacillus species. It should also be kept in mind that a prokaryotic promoter is unlikely to function in a eukaryotic cell, and vise versa. This is important because in genetic engineering studies, gene transformations from one cell to another often take place. In such studies, if gene expression is required following transformation, the gene should always be placed downstream of a promoter that will be recognized by the RNA-polymerase of the particular cell type. -mRNA stability: Once a mRNA is synthesized it is translated into protein molecules. However, certainly this does not go on forever, as it would be a waste of energy and material. Different mRNAs have different half lives. Some mRNAs are more stable than others, whereas others are very unstable and are rapidly degraded. In the latter case, protein synthesis takes place only as long as transcription continues. -post-translational regulation: The activity of the protein may also be regulated. Certain critical proteins are synthesized in an inactive manner and are only activated when necessary. The heat shock proteins are an example. When the temperature increases this triggers the heat shock response and the metabolism is adjusted to the particular alarm situation. It can be easily suggested that such mechanisms aim at saving time and preventing the risk of not being able to express the genes of heat shock proteins under temperature stress conditions. Thus, although it may sound like a waste of energy to synthesize and keep certain proteins (or other molecules, e.g. some RNAs) in an inactive form, as seen from the above example, this is only the case for critical molecules of vital importance. Activation may be by the action of proteases or nucleases, or by glycosylation or phosphorylation. To summarize, regulation of gene expression can take place at any of the 4 major levels, namely: 1. at the level of transcription (initiation, elongation or termination) 2. post-transcriptionally (e.g. pre-mRNA processing, ribosomal and transfer RNA processing, mRNA stability..etc) 3. at the level of translation (initiation, elongation or termination) 4.post-translationally (protein modifications, interactions with other molecules..etc.)

The primary level of control is at the level of transcription, because this is the way by which time and energy can be saved at most, before an unnecessary protein is synthesized. 2.4 Protein Targeting The result of translation is protein synthesis, however, often proteins undergo a number of further steps before they are ready for their particular cellular function. For example, a protein may become an enzyme acting in the cytoplasm or it may be secreted to the extracellular medium. A protein may be transported to the cell membrane to perform a structural function or to the mitochondria, nucleus...etc. The reason that some enzymes may be sent to the extracellular medium is simply because the substrates of these enzymes are located in the extracellular medium. One example is starch, which is a large molecular weight compound and should be first hydrolysed by enzymes (amylases) before it can pass through the cell wall. The intracellular process of transporting proteins to their actual locations is called 'sorting'. The sorting mechanism is generally based on the presence or absence of some signals on the protein. For example, in the case of extracellular proteins, there is a 'signal peptide' at the Nterminus of the protein, which is recognized as the secretion signal. The signal peptide is cleaved during passage to the endoplasmic reticulum (ER) in eukaryotes and the cytoplasmic membrane in prokaryotes. Thus, the signal peptide does not exist in the mature protein, but it's existance can be traced by looking at the corresponding gene. Passage to the ER is the first step of the secretion process in eukaryotes. This occurs in a co-translational manner; i.e. when translation is initiated on an mRNA, if present, the first sequences to be translated will be those corresponding to the signal peptide. Once the signal peptide is translated, translation is temporarily arrested. This is the signal for secretion, resulting in the transportation of the translation complex (mRNA, tRNAs, signal peptide..etc.) to the ER membrane. Once transportation is complete, the signal peptide passes through the ER membrane where it is cleaved, translation is re-initiated and protein is translated and transported through the ER membrane at the same time (in a co-translational manner) (see figure 14.21 in Russell). No consensus sequence (benzer zellikler, ortak bir dizilimin bulunmas) has been established for signal sequences, however, they share a number properties. The length of a signal peptide ranges from 13-36 amino acid. All signal peptides start with 1-2 basic amino acid residues which is followed by a stretch of 10-15 hydrophobic amino acid residues. This so-called

'hydrophobic core' is followed around 5 polar residues prior to the site of cleavage. At the cleavage site, alanine (A) is most commonly encountered but glycine (G), cysteine (C), threonine (T) and serine (S) may also be present. Below is a typical signal peptide:

M-K-L-L-V-V-V-A-V-I-A-C-M-L-I-G-F-A-D-P-A-S-G-C-K-D............

Cytosolic (intracellular) proteins do not contain N-terminal signal sequences and are, therefore, not secreted. However, in biotechnology, when a particular protein is produced on an industrial scale, it is generally desirable that the protein is extracellular, rather than intracellular. This is because, it is easier to separate extracellular proteins from the cells and they are easier to purify because of the less number of contaminating proteins. By genetic engineering, it is possible to convert an intracellular protein to an extracellular form by inserting a signal sequence (that belongs to another protein) upstream (5' end) of the gene. Exercise: 1. Go back to your Biochemistry notes and remember which amino acids are basic, which ones are hydrphobic..etc. 2. Remember the single letter notations for amino acids.

cleavage site

basic residues at the N-terminus

polar C-terminus hydrophobic core