Genetics Chapter 16

Catabolite repression

-(a) In the absence of glucose, cAMP levels increase, resulting in the formation of a CAP-cAMP complex, which binds to the CAP site of the promoter, stimulating transcription -(b) In the presence of glucose, cAMP levels decrease, CAP-cAMP complexes are not formed, and transcription is not stimulated.

Attenuation

-Activated repressor does not inhibit beginning of transcription -When tryptophan concentration is low - attenuation is overcome -ANSWER KEY DEFINITION: functions to reduce the synthesis of tryptophan when it is in full supply. It does so by reducing transcription of the tryptophan operon --the same phenomenon is observed when tryptophan activates the repressor to shut off transcription of the tryptophan operon -can be achieved in a straightforward manner with amino acids, since their availability determines the availibility of corresponding charged tRNAs. It is the availability, or lack there of, those charged tRNAs that regulates transcription

z-y-a genes

-All three genes are transcribed together. Polycistronic mRNA

Cylic adenosine monophosphate (cAMP)

-CAP binding is dependent on this -in order to bind to the promoter, CAP must be bound to _______ -the level of _____ is itself dependent on an enzyme, adenyl cyclase, which catalyzes the conversion of ATP to ____

Operator (one) in Lac

-DNA segment found between the promoter and structural genes. -It determines if transcription will take place. -If the _______ in turned "on", transcription will occur

Regulation

-Does not involve lactose-enzyme interaction (IPTG) -Mutation can also turn on enzymes (Constituitive) (lac I -) -Inducible -all three genes are transcribed by a single unit, resulting in a so-called polycistronic mRNA

Positive Regulation of Lac

-E. coli prefers glucose - maximum energy for growth. -Will only metabolize lactose if concentrations of glucose are low. -Need a signal to tell lac operon that glucose is not available and to start transcribing the genes for lactose metabolism --This signal is cyclic AMP (cAMP)

Trp Operon

-Five Structural Genes: --These genes contain the genetic code for the five enzymes in the trp synthesis pathway -One Promoter: --DNA segment where RNA polymerase binds and starts transcription -One Operator: --DNA segment found between the promoter and structural genes. It determines if transcription will take place. If nothing binds, the operator is turned "on", and transcription will occur -The operon is turned "off" by repressor protein, a product of a repressor gene found some distance from the operon. --Transcription and translation of repressor gene forms an inactive repressor protein that cannot bind to the operator -To become active and bind to the operator, a co-repressor must associate with the repressor. --The co--repressor for this system is Trp -An active repressor binds to the operator blocking the attachment of RNA polymerase to the promoter. -Without RNA polymerase, transcription and translation of the structural genes can't occur and enzymes for Trp synthesis are not made. -this repressible system is under negative control -tryptophan is a corepresor

Attenuation and low trp Levels

-If cellular trp levels are low, translation of the short peptide on domain 1 is slow. -Slow translation causes domain 2 to associate with domain 3, not a ribosome. -This structure allows continued transcription of the operon, translation of the mRNA for the trpE-A genes, and the biosynthesis of tryptophan

Attenuation and high trp levels

-If levels of tryptophan are high, levels of tryptophan tRNA are also high. Immediately after transcription, the small peptide is translated. -Translation is quick because of the high levels of tryptophan tRNA. -Because of the rapid translation, domain 2 becomes associated with the ribosome complex. -Then domain 3 binds with domain 4, and transcription is attenuated because of stem and loop formation

Genetic Proof of Lac Operon model

-Testable Predictions of operon model: --I (repressor gene) encodes a diffusible product --O (operator) regulates transcription, no diffusible product --O region must be next to structural genes

Positive and Negative Control in Lac

-The lac operon is an example of negative genetic control because the operon is shut "off" by a repressor. --Activity restored by removing repressor -Gene regulation would be positive if an activator molecule turned the operon "on". --Activity not initiated until activator binds --The lac operon is also an example of a positive control system and is turned on by a molecular complex (cAMP-CAP)

Structural Genes (three) in Lac

-These encode the three enzymes in the lac catabolic pathway -they code for the primary structure of the enzyme -includes lac z, lac y, and lac a

Allolactose

-When lactose enters the cell, _______, an isomer of lactose is formed. -________ binds to the repressor and alters its conformation so that it can't bind to the operator. --RNA polymerase can now start transcription. -it is an inducer

operon model

-a group of genes is regulated and expressed together as a unit

Cis-acting site

-b/c the regulatory region is on the same strand as those genes, we refer to it as ________

Inducible enzymes

-bacteria adapt to their environment producing certain enzymes only when specific chemical substrates are present

Lac A

-codes for the enzyme transacetylase

Lac I (repressor gene)

-encodes a diffusible product -a constitutive mutation

Lac z

-encodes for beta galactosidase, an enzyme whose primary roe is to convert the disaccharide lactose to the monosaccharides glucose and galactose --this conversion is essential if lactose is to serve as the primary energy source in glycolysis

Constitutive mutation

-enzymes are produced regardless of the presence or absence of lactose

Constitutive Enzymes

-enzymes that are produced continuously, regardless of the chemical makeup of the environment

Negative control

-genetic expression occurs unless it is shut off by some form of a regular molecule

Presence of Glucose

-has a role in catabolite repression -inhibits the activity of adenyl cyclase, causing a decline in the level of cAMP in the cell -under this condition, CAP cannot form the cAMP-CAP complex essential to the positive control of transcription of the lac operon

Regulatory Region

-in prokaryotes, genes that code for enzymes with related functions tend to be organized in clusters on the bacterial chromosome, and transcription of the genes is often under the coordinated control of a single __________ -the location is almost always upstream (5') of the gene cluster it controls -determine whether the genes are transcribed into mRNA and thus whether the corresponding enzymes or other protein products may be synthesized from the genetic information of mRNA

Terminator hairpin

-in the presence of excess tryptophan, the mRNA structure that is formed is referred to as _______, and transcription is almost always terminated prematurely, just beyond the attenuator -when adequate trp is present, charged tRNA^(Trp) is present in the cell --as a result, ribosome treanslate these UGG triplets, proceed through attenuator, and allow _______ to form -it signals RNA polymerase to prematurely terminate transcription, and the operon is not transcribed

Catabolite-activating protein (CAP)

-involved in diminishing the expression of the lac operon when glucose is present -in the absence of glucose and under inducible conditions, ______ exerts positive control by binding to the _____ site, facilitating RNA-polymerase binding at the promoter, and thus transcription -therefore, for maximal transcription for the structural genes, the repressor must be bound by lactose, and CAP must be bound to the CAP-binding site

Lac O (operator region)

-regulates transcription, no diffusible product -must be next to structural genes

Allosteric

-repressor is ______ --the molecule reversibly interacts with another molecule, undergoing both a conformational change in 3-D shape and a change in chemical activity

Lac y

-specifies the primary structure of permease, an enzyme that facilitates the entry of lactose into the bacterial cell

Leader sequence

-that portion of an mRNA molecule from the 5' end to the initiating codon, often containing regulatory or ribosome binding sites --an attenuator was found within this

Antiterminator hairpin

-the initial DNA sequence that is transcribed gives rise to an mRNA sequence that has the potential to fold into two mutually exclusive stem-loop structures referred to as "hairpins" -if tryptophan is scarce, an mRNA secondary structured referred to as the ___________ is formed -transcription proceeds past the __________ region, and the entire mRNA is subsequently produced --the transcript of the leader sequence in the 5'-untranslated region (5'-UTR) of the mRNA must be translated in oder for the ______ to form

Repressible system

-the presence of a specific molecule inhibits gene expression -Tryptophan - If present in medium, enzymes for synthesis are shut off, or repressed

Lac Operon

-three components: --Three Structural Genes: ---These encode the three enzymes in the lac catabolic pathway -One Promoter: --DNA segment where RNA polymerase binds and starts transcription -One Operator: --DNA segment found between the promoter and structural genes. --It determines if transcription will take place. --If the operator in turned "on", transcription will occur -The Lac operon is turned "off" by a specific protein called the repressor. -The repressor is the product of the repressor gene which is found outside the operon. -The repressor is active and does not require a co-repressor. -The active repressor binds to the operator blocking the advancement of RNA polymerase to the structural genes. -Without RNA polymerase, transcription and translation of the genes can't occur and the enzymes needed for lactose metabolism are not made. -Lactose turns on the lac operon. -This makes sense because the cell only needs to make enzymes to catabolize lactose if lactose is present. -When lactose enters the cell, allolactose, an isomer of lactose is formed. -Allolactose binds to the repressor and alters its conformation so that it can't bind to the operator. -RNA polymerase can now start transcription. -The three structural genes are transcribed and the mRNA is translated into the enzymes that control lactose catabolism. -In this sense, allolactose is an inducer.

Positive control

-transcription occurs only if a regulator molecule directly stimulates RNA production

Attenuation of trp operon

-when tryptophan is present and the trp operion is repressed, initiation of transcription still occurs at a low level, but is subsequently terminated at a point 140 nucleotides along the transcript -it weakens or impairs expression of the operon -trp operon contains a leader sequence (L) 5' to the trpE gene. This sequence (~160 bp) controls the operon via a process called attentuation. -This L sequence has four domains (1-4). Domain 3 (nucleotides 108-121) of the mRNA can base pair with either domain 2 (nucleotides 74-94) or domain 4 (nucleotides 126-134). -Domain 1 is also an important component of the attenuation process. The section of the leader sequence encodes a 14 amino acid peptide that has two tryptophan residues.

Trans-acting elements

Cis-acting regulatory regions bind molecules that control transcription of gene cluster

Promoter (one) in Lac

DNA segment where RNA polymerase binds and starts transcription

Inducible system

-Genes for lactose metabolism off. When lactose is present, genes are turned on. Lactose is an Inducer -Three genes are inducible by lactose --lac z (β-gal enzyme) --lac y (permease) --lac a (transacetylase) -Three genes are closely linked --z-y-a is gene order