Midterm 3 Notes PDF

Here are the notes from the provided sources: - **Prokaryotic Transcription** - **Transcription** is the DNA-templated synthesis of RNA. The immediate product of transcription is called the primary transcript. In prokaryotes, the primary transcript is used directly....

Here are the notes from the provided sources: - **Prokaryotic Transcription** - **Transcription** is the DNA-templated synthesis of RNA. The immediate product of transcription is called the primary transcript. In prokaryotes, the primary transcript is used directly. In eukaryotes, pre-mRNA is processed to become mRNA and primary transcripts may be cleaved into tRNA or rRNA. - During **RNA synthesis**, a ribonucleotide is added from an rNTP to the growing transcript through formation of a phosphodiester bond. RNA synthesis occurs in a 5\'→3\' direction. - The enzyme **RNA polymerase** catalyzes RNA synthesis. *E. coli* RNA polymerase consists of several polypeptide subunits. The **σ subunit** can be separated from the core enzyme, which consists of a β subunit, β\' subunit, two α subunits, and an ω subunit. Together, the core enzyme and σ subunit form a **holoenzyme**. There are different σ subunits that can associate with the core enzyme; **σ70 is the most common in *E. coli***. - The presence of the σ subunit allows recognition of **promoters**, which are RNA polymerase binding sites. Transcription that begins at promoters is specific and directed by the σ subunit. - **Sigma factors and promoters** can be studied using a **filter binding assay**. In this assay, radioactive T7 DNA is mixed with RNA polymerase and allowed to bind. Excess non-radioactive T7 DNA is added to compete with the radioactive DNA for binding to the polymerase. The amount of radioactive DNA that remains bound to the polymerase is then measured over time. This assay can be used to determine how tightly core enzyme versus holoenzyme bind DNA. - As temperature is lowered, the binding of RNA polymerase (holoenzyme) to DNA decreases dramatically. Higher temperature promotes tighter DNA binding because it promotes DNA melting. - **Holoenzyme** transiently binds DNA until it finds a promoter; it \"scans\" along the DNA. Holoenzyme initially binds promoter DNA loosely, forming a **closed promoter complex**, in which the DNA is in double-stranded form. Holoenzyme then melts the DNA at the promoter, forming an **open promoter complex**, in which the polymerase is tightly bound. The σ factor is involved in tight binding and transcription initiation. - **Promoters** are sequences of DNA recognized by RNA polymerase holoenzyme. **Core promoter elements**are recognized by the σ subunit. The **-10 region** is 10 bp upstream of the transcription start site, and the **-35 region** is 35 bp upstream of the transcription start site. **UP elements**, found on some promoters of highly expressed genes, are bound by the α subunit. The core promoter elements at -10 and -35 regions are necessary for recognition by the holoenzyme. Promoters recognized by σ70 are not identical, but have a consensus sequence---**TATAAT for the -10 region (Pribnow box) and TTGACA for the -35 region**. - **Mutations** that weaken promoter binding by RNA polymerase are called **down mutations**, which usually increase deviation from the consensus sequence. Mutations that strengthen promoter binding are called **up mutations**, which usually decrease deviation from the consensus sequence. - **DNase footprinting** can be used to analyze protein-DNA interactions and identify where sigma factors bind. Sites at which protein binds DNA are protected from digestion by DNase. In DNase footprinting, one end of a piece of DNA is labeled, the protein is bound, and the complex is treated with DNase. The position where protein binds can be inferred by measuring the length of labeled fragments produced. - Different sigma factors recognize different consensus sequences in the core promoter elements. This has been determined experimentally by protein-DNA interaction studies (DNase footprinting). Different sigma factors may dominate in the cell under different conditions. By using different σ subunits, the cell can coordinate expression of sets of genes, permitting major changes in cell physiology. - In some cases, the consensus sequence for the promoter -10 and -35 elements is not strong enough for the level of gene expression needed. In these cases, an **upstream promoter element (UP element)** can stimulate transcription. The UP element is bound by the α-subunit of RNA polymerase. - **Transcription** occurs in three stages: **initiation, elongation, and termination**. - **Initiation**: During transcription initiation, the σ factor causes tight binding between RNA polymerase and promoters. Tight binding depends on local melting of DNA that permits open promoter complex formation. The σ factor dissociates from the core after enabling polymerase-promoter binding. Transcription initiation involves formation of a closed promoter complex, followed by melting of the DNA to form an open promoter complex. The polymerase then polymerizes a few nucleotides, but remains stuck at the promoter (**abortive transcription**). Eventually, the transcript becomes long enough to form a stable hybrid with the template, and the polymerase clears the promoter. During abortive transcription, the *E.coli* polymerase \"scrunches\" downstream DNA into the polymerase without moving or losing its grip on the promoter. The scrunched DNA stores energy that allows the polymerase to break its bonds to the promoter and begin productive transcription. The σ factor can be recycled for additional use in a process called the **σ cycle**. The σ factor is released from the holoenzyme, and the released σ factor can associate with another core enzyme. In addition to the primary σ-factor, bacteria have alternative σ-factors that allow expression of specialized genes by directing holoenzyme to distinct promoters. - **Elongation**: After transcription initiation is accomplished and RNA polymerase escapes the promoter, the core enzyme continues to elongate the RNA. The σ factor dissociates and is reused for initiation. Nucleotides are added sequentially in the 5\'→3\' direction. RNA polymerase is processive, meaning it is not released until transcription is finished. The core polymerase contains the RNA synthesizing machinery. Phosphodiester bond formation involves the β- and β'-subunits. These subunits also participate in DNA binding. Assembly of the core polymerase is a major role of the α-subunit. During elongation, \~17 bp of DNA duplex is unwound, forming a **transcription bubble** that enables RNA polymerase to access the template strand. The unwinding causes positive supercoils ahead of the polymerase and negative supercoils behind the polymerase. These supercoils are removed by topoisomerases. Elongation involves polymerization of nucleotides as RNA polymerase travels along the template DNA. The polymerase maintains a short, melted region of template DNA. The DNA must unwind ahead of the advancing polymerase and close up behind it. Strain introduced into the template DNA is relaxed by topoisomerases. - **Termination**: There are two main types of terminators: **intrinsic terminators** and **ρ-dependent terminators**. Intrinsic terminators function with RNA polymerase alone, without help from other proteins. ρ-dependent terminators require an auxiliary factor called Rho (ρ). **Intrinsic or rho-independent termination** depends on terminators with two elements: an **inverted repeat** followed immediately by a **T-rich region** in the nontemplate strand of the gene. The inverted repeat predisposes a transcript to form a **hairpin structure**. A string of incorporated U\'s causes RNA polymerase to pause. If a string of U\'s are incorporated just downstream of the hairpin, transcription is terminated. In **rho-dependent termination**, Rho (ρ) binds to the transcript when it lengthens to include the **rho-utilization site (rut)** site. Rho actively feeds the RNA through itself (driven by ATP-hydrolysis). Rho migrates 5′→3′ along the mRNA to the polymerase and causes separation of the mRNA from the polymerase. - **Operons** - Organisms have evolved to turn on expression of genes only when they are needed. Bacterial genes that need to be expressed under the same set of conditions are often grouped together in **operons**, which are regulated together. The **lac operon is the classic example** of an operon. - **Diauxic growth** is the phenomenon in which bacteria grown in a medium containing two different sugars will metabolize the preferred sugar first, then switch to the second sugar after the first is depleted. The **lag phase** between the growth on the two sugars corresponds to the time needed to express the genes necessary for metabolism of the second sugar. - *E. coli* cells need to hydrolyze lactose into monosaccharides to metabolize it. Hydrolysis of lactose is catalyzed by **β-galactosidase**, the expression of which is regulated. - The **lacZ** gene encodes β-galactosidase (LacZ), which needs to be expressed along with **lacY and lacA** in order to metabolize lactose. - LacZ hydrolyzes lactose into monosaccharides. LacZ also isomerizes lactose to allolactose. - The *lacZ, lacY*, and *lacA* genes are normally expressed at low levels, but under appropriate conditions, they can become highly expressed. This occurs when cells sense an **inducer molecule**. - *lacY* and *lacA* encode **galactoside permease** (LacY) and **galactoside transacetylase**, respectively. Permease allows for the uptake of galactosides like lactose. The biological role of transacetylase is not clear. - *lacZ, lacY*, and *lacA* are all under the control of the same promoter, forming an operon. Expression of these genes is turned on in response to an appropriate molecule being sensed. Upstream of the *lac* operon is the *lacI*gene, which codes for the **Lac repressor** and has its own promoter. - The **Lac repressor** is a key component in the negative control of the *lac* operon. The repressor protein is homotetrameric. The repressor protein has two important regions: a domain that responds to the presence of lactose (regulatory domain) and a domain that conditionally binds to DNA (DNA-binding domain). In the absence of an inducer molecule, the *lac* repressor binds to a DNA sequence called an **operator**, and the operon is repressed. The operator sequence is located just downstream of the promoter. RNA polymerase is prevented from transcribing the *lacZ, lacY*, and *lacA* genes. As long as lactose is unavailable, the *lac* operon is repressed. When an **inducer** binds to a repressor, the repressor can no longer bind to the operator, releasing negative control and allowing higher expression of *lacZ, lacY*, and *lacA*. In the *lac* operon, the inducer is **allolactose**, a disaccharide that results from rearrangement of lactose catalyzed by β-galactosidase. β-galactosidase is expressed at low levels without induction. **Isopropyl β-D-thiogalactoside (IPTG)** is a synthetic inducer of the *lac* operon that is not hydrolyzed by β-galactosidase. It is useful in experiments with the *lac* operon. - A **trans-acting genetic element** can influence the expression of a second gene even when the second gene is on a separate chromosome. Trans-acting elements produce a diffusible product. Examples include *lacI* and genes coding for sigma factors. A **cis-acting element** can only influence the expression of a gene that is on the same chromosome. Cis-acting elements do not produce a diffusible product. Examples include promoter regions (-10, -35, and UP elements) and the *lac* operator. - *E. coli* is a monoploid prokaryotic organism with one chromosome. **Merodiploids (partial diploids)** can be generated by transforming *E. coli* with a plasmid encoding its own *lac* operon. This results in one *lac* operon on the chromosome and one on the F\' plasmid. This was done in the **PaJaMo experiment** performed by Arthur Pardee, Francois Jacob, and Jacques Monod. - Genetic analysis of **constitutive mutants** in merodiploids can be used to distinguish between cis- and trans-acting elements. A *lacI*- mutant does not code for a functional repressor, while a *lacO*c mutant has an operator sequence that the repressor protein cannot bind. These regulatory mutations (*I-* and *Oc*) are simple loss-of-function mutations. - Binding of the **Lac repressor** to the *lac* operator DNA sequence can be analyzed by different techniques. A filter-binding assay can be used to show that the wild-type repressor does not bind to the operator when the inducer is present. The Lac repressor binds to an *Oc* operator with lower affinity. An **electrophoretic mobility shift assay (EMSA)** can also be used to study the binding of Lac repressor to the *lac* operator. EMSA observes a shift in the distance traveled by DNA when bound to a protein, because the complex migrates more slowly during electrophoresis. - There are **three *lac* operators**: *O1, O2*, and *O3*. Binding of the Lac repressor to operators prevents binding of RNA polymerase to the promoter. In addition to the major *lac* operator (*O1*), which is adjacent to the promoter, there are two auxiliary *lac* operators (*O2* and *O3*) located upstream (-82) and downstream (+412). All three operators are important for repression. The Lac repressor is a tetramer formed from two homodimers. Each homodimer can bind one operator sequence. Co-ordinate binding of the Lac repressor to *O1* and either *O2* or *O3* leads to a 1000-fold reduction in the rate of transcription. - Negative control of the *lac* operon is released when lactose is present (via induction by allolactose). However, the presence of lactose does not result in high expression of *lacZ, lacY*, and *lacA* if glucose is present. A signal for low glucose is necessary to regulate expression of the *lac* operon. **Cyclic adenosine monophosphate (cAMP)** signals low glucose. - **Positive control** of the *lac* operon involves three factors: - - - - Binding of the CRP-cAMP complex to a DNA sequence upstream of the promoter (the activator-binding site) helps to recruit RNA polymerase to the promoter sequence. - Conversion of AMP to cAMP is catalyzed by **adenylate cyclase**, the activity of which is inhibited by glucose. Thus, cAMP concentration is low when glucose is present and high when glucose is absent. The *cyaA* gene codes for adenylate cyclase. - When bound to cAMP, the resulting CRP-cAMP homodimeric complex binds to the **CRP-binding site**. RNA polymerase is not recruited effectively by the *lac* promoter alone; recruitment therefore needs the help of CRP-cAMP. CRP-cAMP binds DNA and also binds RNA polymerase. CRP-cAMP stimulates formation of the open promoter complex, resulting in high levels of transcription. CRP/CRP-cAMP is also involved in positive regulation of genes required for metabolism of other sugars, including arabinose and galactose. - **Arabinose** is a 5-carbon sugar that can be utilized by *E. coli*. The metabolic enzymes necessary for arabinose utilization are coded by the *araA, araB*, and *araD* genes and include L-arabinose isomerase, ribulokinase, and L-ribulose-5-phosphate 4-epimerase. These genes are grouped together in the **ara operon** under the control of the *ara* promoter. - The **ara operon** is a set of genes that are coordinately regulated. Arabinose is an alternative carbon/energy source, and its utilization is positively regulated by CRP-cAMP. Another protein, **AraC**, which is both a negative and a positive regulator, also regulates the *arabinose* operon. Whether AraC acts as an activator or a repressor is controlled by arabinose. - Expression of *araA, araB*, and *araD* in the *ara* operon is driven by the *araPBAD* promoter. A repressor protein, **AraC**, is involved in negative regulation. In the absence of arabinose, dimeric AraC repressor binds to the *araO2* operator site and *araI1*. Each subunit binds to either DNA site. Binding of the distant *araO2* and *araI1* sites results in a DNA loop that prevents binding of RNA polymerase. Arabinose is an inducer that binds AraC, causing it to bind *araI1* and *araI2* and unbind *araO2*, opening the DNA loop (positive control). Positive control by CAP-cAMP may also involve looping. - **Galactose** is another alternative carbon/energy source to glucose. Genes required for galactose metabolism are in the **gal operon**. The **Gal repressor** exerts negative control on the *gal* operon differently from the Lac repressor. It does not prevent RNA polymerase from binding the promoter, but instead prevents the transition from the closed complex to the open complex, blocking formation of the elongation-competent form of RNA polymerase. - The *E. coli* **trp operon** contains genes for the enzymes that the bacterium needs to make the amino acid tryptophan. The *trp* operon codes for **anabolic enzymes**, those that build up a substance. Anabolic enzymes are typically turned off by a high level of the substance produced. Negative control of the *trp* operon occurs when tryptophan is present in high concentrations. The *trpR* gene encodes an \"**aporepressor**\" that binds tryptophan, which acts as a **co-repressor**, to form the repressor. Binding of the repressor to operator *trpO* is conditional upon tryptophan and prevents transcription of *trp* genes. Unlike the Lac, Ara, and Gal repressors, binding of the effector molecule to the Trp repressor results in a conformation that binds DNA. - The level of tryptophan operon expression in the absence of tryptophan in the medium is about 700 times higher than when tryptophan is plentiful. The tryptophan repressor accounts for about 70-fold of the difference, while the remainder of the regulation, about 10-fold, is due to **attenuation**. **Attenuation** is the termination of *trp* operon transcription before the polymerase reaches the *E, D, C, B*, and *A* genes that code for the enzymes that catalyze tryptophan biosynthesis. It imposes an extra level of control on the operon and operates by causing premature termination of the operon\'s transcript when the product is abundant. - The mRNA transcript of the *trp* operon can form a stable **double-hairpin secondary structure** that includes part of the region coding the leader peptide. The double-hairpin contains codons for two tandem tryptophans (2 × UGG) and a stop codon (UGA). The second hairpin, followed by a string of U\'s in the transcript, promotes **intrinsic termination**. An alternative, less stable secondary structure consists of a single hairpin. The single hairpin is remote enough from the string of Us that it does not promote termination. - The leader peptide encoded by *trpL* contains two tryptophans. It is rare for two tryptophan residues to be found in tandem in a protein/peptide. The presence of two Trp codons in the sequence that can form a double-hairpin is important for sensing the levels of tryptophan in the cell. In conditions of low tryptophan, the ribosome will stall at the tandem Trp (UGG) codons because tryptophanyl-tRNA is not readily available, slowing translation at these codons. The ribosome bound to the tandem Trp codons prevents formation of the double-hairpin RNA structure, disfavoring termination. If the ribosome can translate through the Trp codons swiftly, this allows the double hairpin to form, favoring termination of the *trp* leader mRNA. - Attenuation is a possible mechanism for transcriptional regulation because in prokaryotes, transcription and translation occur concurrently in the cytoplasm, which is not true for eukaryotes. Translation begins as mRNA is being elongated, even before termination and release of the transcript. - A **riboswitch** is an RNA structure (usually found in the 5\'-UTR of mRNA) that, upon binding a ligand, changes conformation and alters expression of the mRNA. Riboswitches consist of a small-molecule-binding element connected to a regulatory region. Expression is controlled by highly specific binding of the small-molecule-binding element to its target ligand, which triggers a conformational change in the regulatory region, altering the structure of the RNA. This can affect transcription or translation. - **Global Changes in Bacterial Transcription** - RNA polymerase holoenzyme requires a **σ-factor** to recognize genes to be transcribed. The σ-factor recognizes -10 and -35 promoter elements (sequences similar to a consensus sequence). Different σ-factors recognize different consensus sequences. **Time dependent programming of gene expression can result from switching the σ-factor of the holoenzyme.** This turns on transcription of multiple genes driven by promoters recognized by the new σ-factor and turns off transcription of genes driven by promoters recognized by the replaced σ-factor. Sigma factor switching is observed during infection of bacteria by some phage and in bacterial sporulation. Examples include T4 phage infection in *E. coli*, SPO1 infection in *B. subtilis*, and formation of endospores in *B. subtilis*. - Phage **SPO1** has a very large genome (145 kbp, \~200 genes) and exhibits a time-dependent programmed gene transcription pattern upon infection of *B. subtilis*. Time-dependent (temporal) control of transcription is divided into three phases: expression of early genes (first 5 minutes), expression of middle genes (5--10 minutes after infection), and expression of late genes (10 minutes after infection). In each phase, an RNA polymerase holoenzyme with a different σ-factor is used, recognizing three different classes of promoter. Early genes (including gene 28) are transcribed using the host σ-factor. Middle genes (including genes 33 and 34) are transcribed using the phage-encoded σ-factor gp28 (encoded by gene 28). Late genes are transcribed using the phage-encoded σ-factor consisting of subunits gp33 and gp34 (encoded by genes 33 and 34). - The *B. subtilis* genome codes for at least 18 different **sigma factors**. Each sigma recognizes a specific promoter sequence. The regulated expression of different sigmas enables major shifts in gene expression. *B. subtilis* **sporulation** involves switching on sporulation genes and switching off many vegetative genes. This reprogramming happens by changing sigma factors. Multiple sigma factors are involved in global gene expression during sporulation, including σF, σE, σH, σC, and σK. These new sigma factors, which are expressed during sporulation, act together with σA, which is the main sigma factor of the vegetative state. - During active growth of *E. coli*, the majority of the transcriptional activity is carried out by the housekeeping sigma factor, σ70. In stationary phase, under starvation conditions, or under stress, alternative sigma factors drive transcription of genes to respond to the stress. These include σ54, σ38, σ32, and σ24. - **Heat shock** (e.g., shifting temperature from 37°C to 42°C) causes many native *E. coli* proteins to denature (unfold to varying degrees). To deal with heat shock, *E. coli* transiently turns on expression of about 17 genes (the **heat shock genes**). Heat shock genes code for molecular chaperones and proteases. Chaperones bind to misfolded proteins and help them to fold properly, while proteases degrade proteins that are too badly denatured. Heat shock proteins are regulated by the alternative sigma factor **σ32 (RpoH)**. - The heat-shock response is fast (begins in less than a minute) because σ32 (RpoH) is already present prior to heat-shock. At normal temperatures, σ32 is bound by heat shock proteins, which destabilize and eventually destroy it, preventing its function. At elevated temperatures, heat-shock proteins bind other denatured proteins, freeing up σ32 to perform its function. Additionally, the 5′ region of the mRNA coding for σ32 can form a stem loop structure that blocks binding of the ribosome to the ribosomal binding site. High temperatures denature the stem, thereby increasing σ32 translation. - Nitrogen starvation and entry into stationary phase switch on expression of **σ54 (RpoN)** and **σ38 (RpoS)**, respectively. **Anti-sigma-factors** also play a role in regulating sigma factor activity. In response to starvation, high temperature, and high osmolarity, *E. coli* makes **Rsd (Regulator of sigma D)**. Rsd binds to **σ70 (RpoD, major vegetative sigma factor)** and prevents it from binding to housekeeping promoters. This lowers global expression levels to conserve energy and resources when they are scarce. - The **T7 phage**, a bacteriophage that infects *E. coli*, has three phases of transcription: early phase (class I), and two late phases (class II and III). The host RNA polymerase, along with host σ70 (RpoD), transcribes class I early phase genes (5 in total). One of the class I genes encodes the T7 polymerase. The phage T7 polymerase then transcribes class II and III genes. - Phage **λ** infects *E. coli* and regulates programmed gene expression using anti-termination. λ is a \"temperate\" phage, meaning it does not always kill the host cell it infects. It can follow either of two pathways: **lysogeny or lysis**. The survival strategy of phage λ is unlike that of phages T2, T4, T7, and SPO1, which are \"virulent\" phages---they always replicate and kill their hosts by lysis. - **Bacteriophage lambda** can enter either the lytic or lysogenic cycle upon infection of *Escherichia coli*. - λ DNA exists in linear form in the phage. After infection, the phage DNA circularizes. This is possible because the linear form has **sticky ends (cohesive sites, cos)**. Gene transcription is controlled by transcriptional switches, including repressors, activators of transcription, and antitermination. - **N protein** is an antiterminator. Its utilization requires the *nut* (*N* utilization) site on the RNA transcript. When N is complexed to the transcript during elongation, the termination signal is ignored, allowing downstream genes to be transcribed. *In vitro* studies show that antitermination requires assembly of multiple host proteins together with protein N in a complex at the *nut* site. N-dependent antitermination switches the gene expression program from immediate early to delayed early. - **Q protein** is another antiterminator and is a delayed early gene. Q-dependent antitermination is responsible for switching from the delayed early program to the late program. Unlike N protein, antiterminator Q binds to DNA at the **Q-binding site (qut)**, not the transcript. In the absence of antiterminator Q, RNA polymerase stalls and then aborts at a pause site. When Q is present, it recognizes the paused complex, binds at *qut*, and interacts with the polymerase, allowing it to continue past the pause site. Antitermination in the λ late region requires Q. Polymerase associated with Q can ignore the termination signal just downstream of *PR\'*. Binding of Q to the polymerase appears to alter the enzyme so that it can ignore the terminator and transcribe the late genes. - **CII** is a delayed early gene product that acts as an activator of *PRE*-driven *CI* expression. **CIII** protects CII from enzymatic proteolysis. - The *cI* gene encodes a 27-kD phage protein (**λ repressor, CI**). CI binds to two phage operator regions and shuts down transcription of all genes except for *cI*, the gene encoding itself. CI function counteracts that of **Cro**, another repressor that represses expression of *cI*. - When lysogeny is established, the phage DNA integrates into the bacterial genome. A bacterium harboring integrated phage DNA is called a **lysogen**, and the integrated DNA is called a **prophage**. The phage DNA in the lysogen replicates along with the host DNA. - The **lytic mode of reproduction** results in viral replication and rupture of host cells, releasing phage particles. The lytic reproduction cycle of λ phage has three phases of transcription: immediate early, delayed early, and late. The time-dependent transcription pattern is controlled largely by antitermination. Genes of these phases are arranged sequentially on the phage DNA. - The λ gene expression program involves repressors and activators of transcription, along with anti-termination. The program uses conditional transcription termination signals. The transcript is limited to early genes in the first few minutes of infection. The pattern of gene expression is changed by overriding these conditional termination signals (anti-termination). - Transcription in λ phage begins with the immediate early genes from the *PL* and *PR* promoters. Transcription stops at the terminator downstream of the *N* gene (on the left side) and downstream of the *cro* gene (on the right side). The expression of *N* allows subsequent transcripts to bypass these terminators. - If immediate early, delayed early, and late gene expression all occur, the phage proceeds to the **lytic cycle**. This results in phage genome replication, phage structural protein production, assembly of phage particles, production of cell-lysing proteins, cell lysis, and release of progeny phage. - Alternatively, **lysogeny** can occur, in which case the infected *Escherichia coli* survives. The order of events during lysogeny is as follows: - - - - - - - The order of events during establishment of lysogeny is as follows: - - - - The λ repressor, \*\*