Lec 3-1: The Lac Operon of E. coli PDF

The lac Operon of E. coli Lactose metabolism Lactose is a major carbohydrate found in milk; it can be metabolized by E. coli bacteria that reside in the mammalian gut. Lactose does not easily diffuse across the E. coli cell membrane and must be actively transported into the cell by the prote...

The lac Operon of E. coli Lactose metabolism Lactose is a major carbohydrate found in milk; it can be metabolized by E. coli bacteria that reside in the mammalian gut. Lactose does not easily diffuse across the E. coli cell membrane and must be actively transported into the cell by the protein permease. To utilize lactose as an energy source, E. coli must first break it into glucose and galactose, a reaction catalyzed by the enzyme β-galactosidase. β-galactosidase can also convert lactose into allolactose, a compound that plays an important role in regulating lactose metabolism. A third enzyme, thiogalactoside transacetylase, also is produced by the lac operon, but its function in lactose metabolism is not yet known. Regulation of the lac operon The lac operon is an example of a negative inducible operon. The enzymes β- galactosidase, permease, and transacetylase are encoded by adjacent structural genes in the lac operon of E. coli and have a common promoter (lacP). β-Galactosidase is encoded by the lacZ gene, permease by the lacY gene, and transacetylase by the lacA gene. If lactose is added to the medium and glucose is absent, the rate of synthesis of all three proteins simultaneously increases about a thousand fold within 2 to 3 minutes. This boost in protein synthesis results from the transcription of lacZ, lacY, and lacA and exemplifies coordinate induction, the 1 simultaneous synthesis of several proteins, stimulated by a specific molecule, the inducer. Although lactose appears to be the inducer here, allolactose is actually responsible for induction. Upstream of lacP is a regulator gene, lacI with its own promoter (PI). The lacI gene is transcribed into mRNA that is translated into a repressor. The repressor has two types of binding sites; one type of 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 lacO that actually overlaps the 3′ end of the promoter and the 5′ end of lacZ. Thus, when the repressor is bound to the operator, 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 lacZ, lacY, and lacA takes place, and the lac proteins are produced. The trp Operon of E. coli The tryptophan (trp) operon in E. coli, which controls the biosynthesis of the amino acid tryptophan, is an example of a negative repressible operon. Transcription is normally turned on and must be repressed. 2 The trp operon contains five structural genes (trpE, trpD, trpC, trpB, and trpA) that produce the components of three enzymes (two of the enzymes consist of two polypeptide chains). These enzymes convert chorismate into tryptophan. Some distance from the trp operon is a regulator gene, trpR, which encodes a repressor that alone cannot bind DNA. The tryptophan repressor has two binding sites, one that binds to DNA at the operator site and another that binds to tryptophan (the activator). Binding with tryptophan causes a conformational change in the repressor that makes it capable of binding to DNA at the operator site, which overlaps the promoter. When the operator is occupied by the tryptophan repressor, RNA polymerase cannot bind to the promoter and the structural genes cannot be transcribed. Thus, when cellular levels of tryptophan are low, transcription of the trp operon takes place and more tryptophan is synthesized; when cellular levels of tryptophan are high, transcription of the trp operon is inhibited and the synthesis of more tryptophan does not take place. Some Operons Regulate Transcription Through Attenuation, the Premature Termination of Transcription We’ve now seen several different ways in which a cell regulates the initiation of transcription in an operon. Some operons have an additional level of control that affects the continuation of transcription rather than its initiation. In attenuation, transcription begins at the start site, but termination takes place prematurely, before the RNA polymerase even reaches the structural genes. Attenuation takes place in a number of operons that encode enzymes participating in the biosynthesis of amino acids. We can understand the process of attenuation most easily by looking at one of the best-studied examples, which is found in the trp operon of E. coli. The trp operon is unusual in that it is regulated both by repression and by attenuation. Most operons are regulated by one of these mechanisms but not by both of them. The first structural gene, trpE, contains a long 5′ untranslated region (5′ UTR) that is transcribed but does not encode any of these enzymes. Instead, this 5′ UTR plays an important role in another regulatory mechanism. Upstream of the 5′ UTR is the trp promoter. When tryptophan levels are low, RNA polymerase binds to the promoter and transcribes the five structural genes into a single mRNA, which is then translated into enzymes that convert chorismate into tryptophan. Close examination of the trp operon reveals a region of 162 nucleotides that corresponds to the long 5′ UTR of the mRNA (mentioned earlier) transcribed from 3 the trp operon. The 5′ UTR (also called a leader) contains four regions: region 1 is complementary to region 2, region 2 is complementary to region 3 and region 3 is complementary to region 4. These complementarities allow the 5′ UTR to fold into two different secondary structures. Only one of these secondary structures causes attenuation. One of the secondary structures contains one hairpin produced by the base pairing of regions 1 and 2 and another hairpin produced by the base pairing of regions 3 and 4. Notice that a string of uracil nucleotides follows the 3+4 hairpin. Not coincidentally, the structure of a bacterial intrinsic terminator includes a hairpin followed by a string of uracil nucleotides; this secondary structure in the 5′ UTR of the trp operon is indeed a terminator and is called an attenuator. The attenuator forms when cellular levels of tryptophan are high, causing transcription to be terminated before the trp structural genes can be transcribed. When cellular levels of tryptophan are low, however, the alternative secondary structure of the 5′ UTR is produced by the base pairing of regions 2 and 3. This base pairing also produces a hairpin, but this hairpin is not followed by a string of uracil nucleotides; so this structure does not function as a terminator. RNA polymerase continues past the 5′ UTR into the coding section of the structural genes, and the enzymes that synthesize tryptophan are produced. Because it prevents the termination of transcription, the 2+3 structure is called an antiterminator. 4 Region 1 encodes a small protein. Within the coding sequence for this protein are two UGG codons, which specify the amino acid tryptophan; so tryptophan is required for the translation of this 5′ UTR sequence. The small protein encoded by the 5′ UTR has not been isolated and is presumed to be unstable; its only apparent function is to control attenuation. Transcription when tryptophan levels are low Recall that, in prokaryotic cells, transcription and translation are coupled: Closely following RNA polymerase, a ribosome binds to the 5′ UTR and begins to translate the coding region. Meanwhile, RNA polymerase is transcribing region 2. Region 2 is complementary to region 1 but, because the ribosome is translating region 1, the nucleotides in regions 1 and 2 cannot base pair. RNA polymerase begins to transcribe region 3, and the ribosome reaches the UGG tryptophan codons in region 1. When it reaches the tryptophan codons, the ribosome stalls because the level of tryptophan is low and tRNAs charged with tryptophan are scarce or even unavailable. The ribosome sits at the tryptophan codons, awaiting the arrival of a tRNA charged with tryptophan. Stalling of the ribosome does not, however, hinder transcription; RNA polymerase continues to move along the DNA, and transcription gets ahead of translation. Because the ribosome is stalled at the tryptophan codons in region 1, region 2 is free to base pair with region 3, forming the 2+3 hairpin. This hairpin does not cause termination, and so transcription continues. Because region 3 is already paired with region 2, the 3+4 hairpin (the attenuator) never forms, and so attenuation does not take place and transcription continues. RNA polymerase continues along the DNA, past the 5′ UTR, transcribing 5 all the structural genes into mRNA, which is translated into the enzymes encoded by the trp operon. These enzymes then synthesize more tryptophan. It is important to point out that ribosomes do not traverse the convoluted hairpins of the 5′ UTR to translate the structural genes. Ribosomes that attach to the 5′ end of region 1 of the mRNA encounter a stop codon at the end of region 1. New ribosomes translating the structural genes attach to a different ribosome-binding site located near the beginning of the trpE gene. Transcription when tryptophan levels are high RNA polymerase begins transcribing the DNA, producing region 1 of the 5′ UTR. Closely following RNA polymerase, a ribosome binds to the 5′ UTR and begins to translate the coding region. When the ribosome reaches the two UGG tryptophan codons, it doesn’t slow or stall, because tryptophan is abundant and tRNAs charged with tryptophan are readily available. This point is critical to note: because tryptophan is abundant, translation can keep up with transcription. As it moves past region 1, the ribosome partly covers region 2; meanwhile, RNA polymerase completes the transcription of region 3. Although regions 2 and 3 are complementary, the ribosome physically blocks their pairing. RNA polymerase continues to move along the DNA, eventually transcribing region 4 of the 5′ UTR. Region 4 is complementary to region 3, and, because region 3 cannot base pair with region 2, it pairs with region 4. The pairing of regions 3 and 4 produces theattenuator and transcription terminates just beyond region 4. The 6 structural genes are not transcribed, no tryptophanproducing enzymes are translated, and no additional tryptophan is synthesized. RNA Molecules Control the Expression of Some Bacterial Genes Antisense RNA Some small RNA molecules are complementary to particular sequences on mRNAs and are called antisense RNA. They control gene expression by binding to sequences on mRNA and inhibiting translation. Translational control by antisense RNA is seen in the regulation of the ompF gene of E. coli. ompF gene encodes an outer-membrane protein that functions as a channel for the passive diffusion of small polar molecules, such as water and ions, across the cell membrane. Under most conditions, the ompF gene is transcribed and translated and the OmpF protein is synthesized. However, when the osmolarity of the medium increases, the cell depresses the production of OmpF protein to help maintain cellular osmolarity. A regulator gene named micF—formRNA-interfering complementary RNA—is activated and micF RNA is produced. The micF RNA, an antisense RNA, binds to a complementary sequence in the 5′ UTR of the ompF mRNA and inhibits the binding of the ribosome. This inhibition reduces the amount of translation, which results in fewer OmpF proteins in the outer membrane and thus reduces the detrimental movement of substances across the membrane owing to the changes in osmolarity. 7 Riboswitches Some mRNA molecules contain regulatory sequences called riboswitches, where molecules can bind and affect gene expression by influencing the formation of secondary structures in the mRNA. Riboswitches were first discovered in 2002 and now appear to be common in bacteria, regulating about 4% of all bacterial genes. They are also present in archaea, fungi, and plants. Riboswitches are typically found in the 5′ UTR of the mRNA and fold into compact RNA secondary structures with a base stem and several branching hairpins. In some cases, a small regulatory molecule binds to the riboswitch and stabilizes a terminator, which causes premature termination of transcription. In other cases, the binding of a regulatory molecule stabilizes a secondary structure that masks the ribosome-binding site, preventing the initiation of translation. When not bound by the regulatory molecule, the riboswitch assumes an alternative structure that eliminates the premature terminator or makes the ribosome binding site available. 8  Gene Regulation in Eukaryotes Eukaryotic cells and bacteria have many features of gene regulation in common, but they differ in several important ways in both types of cells, DNA-binding proteins influence the ability of RNA polymerase to initiate transcription. However, there are also some differences. First, most eukaryotic genes are not organized into operons and are rarely transcribed together into a single mRNA molecule, although some operon-like gene clusters have been discovered in eukaryotes. In eukaryotic cells, each structural gene typically has its own promoter and is transcribed separately. Second, chromatin structure affects gene expression in eukaryotic cells; DNA must unwind from the histone proteins before transcription can take place. Third, the presence of the nuclear membrane in eukaryotic cells separates transcription and translation in time and space. Therefore, the regulation of gene expression in eukaryotic cells is characterized by a greater diversity of mechanisms that act at different points in the transfer of information from DNA to protein. Eukaryotic gene regulation is less well understood than bacterial regulation, partly owing to: The larger genomes in eukaryotes, The greater sequence complexity in eukaryotes, The difficulty of isolating and manipulating mutations that can be used in the study of gene regulation. Changes in Chromatin Structure Affect the Expression of Genes One type of gene control in eukaryotic cells is accomplished through the modification of chromatin structure. In the nucleus, histone proteins associate to form octamers, around which helical DNA tightly coils to create chromatin. 9 In a general sense, this chromatin structure represses gene expression. For a gene to be transcribed, transcription factors, activators, and RNA polymerase must bind to the DNA. How can these events take place with DNA wrapped tightly around histone proteins? The answer is that, before transcription, chromatin structure changes and the DNA becomes more accessible to the transcriptional machinery. DNase I Hypersensitivity As genes become transcriptionally active, regions around the genes become highly sensitive to the action of DNase I (when DNA is tightly bound to histone proteins, it is less sensitive to DNase I, whereas unbound DNA is more sensitive to digestion by DNase I) indicating loosening of DNA-histone binding. These regions, called DNase I hypersensitive sites, frequently develop about 1000 nucleotides upstream of the start site of transcription, suggesting that the chromatin in these regions adopts a more open configuration during transcription. This relaxation of the chromatin structure allows regulatory proteins access to binding sites on the DNA. At least three different processes affect gene regulation by altering chromatin structure: The modification of histone proteins; 10 Histones in the octamer core of the nucleosome have two domains: A globular domain that associates with other histones and the DNA A positively charged tail domain that probably interacts with the negatively charged phosphate groups on the backbone of DNA. The tails of histone proteins are often modified by the addition or removal of: phosphate groups, methyl groups, or acetyl groups. These modifications have sometimes been called the histone code, because they encode information that affects how genes are expressed (through altering chromatin packing allowing or preventing binding to transcription machinary). Chromatin remodeling; Some transcription factors and other regulatory proteins alter chromatin structure without altering the chemical structure of the histones directly. These proteins are called chromatin-remodeling complexes. They bind directly to particular sites on DNA and reposition the nucleosomes, allowing transcription factors to bind to promoters and initiate transcription. 11 Evidence suggests at least two mechanisms by which remodeling complexes reposition nucleosomes. First, some remodeling complexes cause the nucleosome to slide along the DNA, allowing DNA that was wrapped around the nucleosome to occupy a position in between nucleosomes, where it is more accessible to proteins affecting gene expression. Second, some complexes cause conformational changes in the DNA, in nucleosomes, or in both so that DNA that is bound to the nucleosome assumes a more exposed configuration. DNA methylation. The methylation of cytosine in DNA is distinct from the methylation of histone proteins mentioned earlier. The methylation of cytosine bases, which yields 5- methylcytosine is associated with change in chromatin structure. Heavily methylated DNA is associated with the repression of transcription in vertebrates and plants, whereas transcriptionally active DNA is usually unmethylated in these organisms. 12 Epigenetic Effects Often Result from Alterations in Chromatin Structure Epigenetics are alterations to DNA and chromatin structure that affect traits and are passed on to other cells or future generations but are not caused by changes in the DNA base sequence. Epigenome is the overall pattern of chromatin modifications possessed by each individual organism. The Initiation of Transcription Is Regulated by Transcription Factors and Transcriptional Regulator Proteins General transcription factors and RNA polymerase assemble into a basal transcription apparatus, which binds to a core promoter located immediately upstream of a gene. The basal transcription apparatus is capable of minimal levels of transcription. Transcriptional regulator 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 other regulatory sequences eg: enhancers or silencers, which may be located some distance from the gene. Some transcriptional regulator proteins are activators, stimulating transcription. Others are represssors, inhibiting transcription. 13 Transcriptional Activators and Coactivators Transcriptional activator proteins stimulate and stabilize the basal transcription apparatus at the core promoter. The activators may interact directly with the basal transcription apparatus or indirectly through protein coactivators. Some activators and coactivators, as well as the general transcription factors, also have acteyltransferase activity and so further stimulate transcription by altering chromatin structure. Transcriptional activator proteins binding to sequences in the regulatory promoter (or enhancer) make contract with the mediator and affect the rate at which transcription is initiated. Regulation of galactose metabolism through GAL4 An example of a transcriptional activator protein is GAL4, which regulates the transcription of several yeast genes whose products metabolize galactose. The genes that control galactose metabolism are inducible: when galactose is absent, these genes are not transcribed and the proteins that break down galactose are not produced; when galactose is present, the genes are transcribed and the enzymes are synthesized. 14 GAL4 contains several zinc fingers and binds to a DNA sequence called UASG (upstream activating sequence for GAL4). UASG exhibits the properties of an enhancer—a regulatory sequence that may be some distance from the regulated gene and is independent of the gene in position and orientation. GAL4 activates the transcription of yeast genes needed for metabolizing galactose. A particular region of GAL4 binds another protein called GAL80, which regulates the activity of GAL4 in the presence of galactose. When galactose is absent, GAL80 binds to GAL4, preventing GAL4 from activating transcription. When galactose is present, however, it binds to another protein called GAL3, which interacts with GAL80, causing a conformational change in GAL80 so that it can no longer bind GAL4. The GAL4 protein is then free to activate the transcription of the genes, whose products metabolize galactose. Transcriptional Repressors These repressors may compete with activators for DNA binding sites. When a site is occupied by an activator, transcription is activated, but, if a repressor occupies that site, there is no activation. Alternatively, a repressor may bind to sites near an activator site and prevent the activator from contacting the basal transcription apparatus. Enhancers and Insulators How can an enhancer affect the initiation of transcription taking place at a promoter that is tens of thousands of base pairs away? In many cases, regulator proteins bind to the enhancer and cause the DNA between the enhancer and the promoter to loop out, bringing the promoter and enhancer close to each other, and so the transcriptional regulator proteins are able to directly interact with the basal transcription apparatus at the core promoter. 15 Most enhancers are capable of stimulating any promoter in their vicinities. Their effects are limited, however, by insulators (also called boundary elements), which are DNA sequences that block or insulate the effect of enhancers in a position-dependent manner. mRNA Editing For example production of apolipoprotein B100 and apolipoprotein B48 in human In liver: The gene for the protein apolipoprotein B100 codes for 4563 amino acid polypeptide. In intestine: An enzyme called adenosine deaminase acts on RNA. It deaminates certain cytosine into uracil converting CAA codon (specifies glutamine) into UAA (stop codon) resulting into a shorter polypeptide (2153) called apolipoprotein B48. 16  Some Genes Are Regulated by RNA Processing and Degradation Alternative splicing In the T-antigen gene, the T-antigen gene of the mammalian virus SV40 is a well studied example of alternative splicing. This gene is capable of encoding two different proteins, the large T and small t antigens. Which of the two proteins is produced depends on which of two alternative 5′ splice sites is used in RNA splicing. The Degradation of RNA The amount of a protein that is synthesized depends on the amount of corresponding mRNA available for translation. The amount of available mRNA, in turn, depends on both the rate of mRNA synthesis and the rate of mRNA degradation. There is great variability in the stability of eukaryotic mRNA: some mRNAs persist for only a few minutes; others last for hours, days, or even months. These variations can result in large differences in the amount of protein that is synthesized. 17 Most eukaryotic cells contain 10 or more types of ribonucleases, and there are several different pathways of mRNA degradation. In one pathway, the 5′ cap is first removed, followed by 5′→3′ removal of nucleotides. A second pathway begins at the 3′ end of the mRNA and removes nucleotides in the 3′→5′ direction. In a third pathway, the mRNA is cleaved at internal sites. Messenger RNA degradation from the 5′ end is most common and begins with the removal of the 5′ cap. This pathway is usually preceded by the shortening of the poly(A) tail. Poly(A)-binding proteins (PABPs) normally bind to the poly(A) tail and contribute to its stability-enhancing effect. Poly(A)-binding proteins (PABPs) normally bind to the poly(A) tail and contribute to its stability-enhancing effect. The presence of these proteins at the 3′ end of the mRNA protects the 5′ cap. When the poly(A) tail has been shortened below a critical limit, the 5′ cap is removed, and nucleases then degrade the mRNA by removing nucleotides from the 5′ end. These observations suggest that the 5′ cap and the 3′ poly(A) tail of eukaryotic mRNA physically interact with each other, most likely by the poly(A) tail bending around so that the PABPs make contact with the 5′ cap. Much of RNA degradation takes place in specialized complexes called P bodies. Recent evidence suggests that P bodies can temporarily store mRNA molecules, which may later be released and translated. Thus, P bodies help control the expression of genes by regulating which RNA molecules are degraded and which are sequestered for later release. RNA degradation facilitated by small interfering RNAs (siRNAs) also may take place within P bodies. RNA Interference Is an ImportantMechanism of Gene Regulation The expression of a number of eukaryotic genes is controlled through RNA interference, also known as RNA silencing and posttranscriptional gene silencing. Recent research suggests that as much as 30% of human genes are regulated by RNA interference. Although many of the details of this mechanism are still being 18 investigated, RNA interference is widespread in eukaryotes, existing in fungi, plants, and animals. This mechanism is also widely used as a powerful technique for artificially regulating gene expression in genetically engineered organisms Small Interfering RNAs and MicroRNAs RNA interference is triggered by microRNAs (miRNAs) and small interfering RNAs (siRNAs), depending on their origin and mode of action. siRNAs or miRNAs that are from 21 to 25 nucleotides in length pair with proteins to form an RNA-induced silencing complex (RISC). The RNA component of RISC then pairs with complementary base sequences of specific mRNA molecules, most often with sequences in the 3′ UTR of the mRNA. Small interfering RNAs tend to base pair perfectly with the mRNAs, whereas miRNAs often form less-than-perfect pairings. Mechanisms of Gene Regulation by RNA Interference Small interfering RNAs and microRNAs regulate gene expression through at least four distinct mechanisms: 1. Cleavage of mRNA, 2. Inhibition of translation, 3. Transcriptional silencing, 4. Degradation of mRNA. RNA cleavage RISCs that contain an siRNA (and some that contain an miRNA) pair with mRNA molecules and cleave the mRNA near the middle of the bound siRNA. 19 This cleavage is sometimes referred to as “Slicer activity.” After cleavage, the mRNA is further degraded. Thus, the presence of siRNAs and miRNAs increase the rate at which mRNAs are broken down and decrease the amount of protein produced. Inhibition of translation Some miRNAs regulate genes by inhibiting the translation of their complementary mRNAs. The exact mechanism by which miRNAs repress translation is not known. Some research suggests that it can inhibit both the initiation step of translation and steps after translation initiation such as those causing premature termination.Many mRNAs have multiple miRNA-binding sites, and translation is most efficiently inhibited when multiple miRNAs are bound to the mRNA. Transcriptional silencing Other siRNAs silence transcription by altering chromatin structure. These siRNAs combine with proteins to form a complex called RITS (for RNA transcriptional silencing), which is analogous to RISC. The siRNA component of RITS then binds to its complementary sequence in DNA or an RNA molecule in the process of being transcribed and represses transcription by attracting enzymes that methylate the tails of histone proteins. The addition of methyl groups to the histones causes them to bind DNA more tightly, restricting the access of proteins and enzymes necessary to carry out transcription (see earlier section on histone modification). Some miRNAs bind to complementary sequences in DNA and attract enzymes that methylate the DNA directly, which also leads to the suppression of transcription (see earlier section on DNA methylation). Slicer-independent degradation of mRNA A final mechanism by which miRNAs regulate gene expression is by triggering the decay of mRNA in a process that does not require Slicer activity. For example, a short lived mRNA with an AU-rich element in its 3′ UTR is degraded by an RNA- silencing mechanism. Researchers have identified an miRNA with a sequence that is complementary to the consensus sequence in the AU-rich element. This miRNA binds to the AU-rich element and, in a way that is not yet fully understood, brings about the degradation of the mRNA in a process that requires Dicer and RISC. 20  Programmed Frameshifting Ribosome changes the reading frame at a specific point within the transcript. It enables ribosome to produce more than one polypeptide from the same transcript. Programmed frameshifting is controled by the tRNA attached to the growing polypeptide. It occurs when a sequence similar to ribosome binding site occurs just after frameshift position. Posttranslational Processing Of Proteins The polypeptide emerging from ribosome is inactive it needs posttranslational processing. Many eukaryotic proteins are extensively modified after translation by the selective cleavage and trimming of amino acids from the ends, by acetylation, or by the addition of phosphate groups, carboxyl groups, methyl groups, or carbohydrates to the protein. These modifications affect the transport, function, and activity of the proteins and have the capacity to affect gene expression. 21