Quiz 16 Genetics

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2. coupled transcription and translation occur under conditions in which the tryptophan concentration is low, then the cells cannot make a sufficient amount of charged trna. the ribosomes pause at the trp codons in the L mrna because it is waiting for charged trna. this pause occuring leaves the ribosome to shield region 1 of the mrna which prevents region 1 hydrogen bond to region 3, therefor because region 3 is already hydrogen bonded to 2, 3-4 stemloop cannot form. transcription termination will not occur and rna polymerase transcribes the rest of the operon which enables the bacterium to make more tryptophan

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3. coupled transcription and translation occur under conditions in which a sufficient amount of tryptophan is present in the cell. In this case, translation of the L mrna progresses to its stop codon where the ribosome pauses. The pausing at the stop codon prevents region 2 from hydrogen bonding with ANY region thereby enabling region 3 to hydrogen bond with 4. This terminates transcription. Keep in mind that the L mrna contains two tryptophan codons, for the ribosome to smoothly progress to the L stop codon, enough charged trna must be available to translate the mrna

Gene Regulation

level of gene expression can vary under different conditions. Benefit is the encoded proteins are produced only when they are required therefor the cell avoids wasting valuable energy making proteins it does not need.

second transcriptional unit involved in genetic regulation is the lac-i gene which is NOT apart of the lac operon

the lac I gene, expressed at low levels, has its own promoter (i pomoter) lac i gene encodes the LAC REPRESSOR, a protein that is important for the regulation of the lac operon. The lac repressor functions as a HOMOTETRAMER - a protein composed of 4 identical subunits. 10 proteins per cell is made. Small amount of the lac repressor protein is needed to repress the lac operon.

interpret the data

Yellow = absence or presence of lactose the presence of lactose was not needed to induce the operon due to a defective lac i gene. merozygote strain - absence of lactose the lac operon were repressed, even the operon on the bacterial chromosome. Why? because the normal lac i gene on the F' factor was not physically located next to the chromosomal lac operon, this result is consistent with the idea that the lac i gene coes for a repressor protein that can diffuse throughout the cell and bind to any lac operon hypothesis that the lac i mutation results in the synthesis of an internal inducer was rejected. If the hypothesis WAS correct then the merozygote strian would have still made an internal inducer and the lac operon in the merozygote would have been expressed in the absence of lactose.

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a mutation in a transacting factor can be complemented by the introduction of a second gene with normal function. a mutation in a cis acting element is not affected by the intro of another cis acting element with a normal funciton into the cell

Regulation of the lac operon

found in e.coli involves genes that play a role in the utilization of lactose which is a sugar found in milk, the regulation of these genes involves a repressor protein and activator protein.

*** The binding of _ to the lac repressor causes the lac repressor to _ o the operator site thereby _ transcription

glu, not bind, increase

lactose metabolism in Ecoli

1. the expose of bacterial cells ot lactose increased levels of lactose-utilizing enzymes by 1000- to 10000-fold 2. anti body and labeling techniques revealed that the increase in the activity of these enzymes was due to increased synthesis of the enzymes 3. the removal of lactose form the environment caused an abrupt termination in the synthesis 4. mutations that prevented the synthesis of particular proteins involved in lactose utilization showed a separate gene encoded each protein

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16.7 lac-i mutant strain was already known to constitutively express the lac operon and compared it to the corresponding merozygote. The merozygote had a lac-i negative mutant gene on the chromosome and a normal lac i gene on an F' factor. These two strains were grown then divided into two tubes. Half of the tubes, lactose was omitted and the other half the strains were incubated with lactose to determine if lactose was needed to induce the expression of the operon. the cells were lyced by sonication and then a lactose analog, betanitrophenulgalactoside (B-ONPG) was added. The amount of yellow color produced in a given amount of time is a measure of the amount of beta-galactosidase that is being expressed from the lac operon.

lac operon is regulated by a repressor protein

Transcription can occur in more than one way This way is inducible and under negative control. this regulation involves the lac repressor protein which binds to the sequence of nucleotides found within the lac operator site. Once bound, the lac repressor prevents RNa polymerase from transcribing the lacZ, Y, and A genes. The binding of the repressor to the operator site is a reversible process. When no allolactose is present, the lac repressor is bound to the operator site.

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Using ttwo sugars by a bacterium = DIAUXIC GROWTH glucose is not the small effector that binds to a genetic regulatory molecule, CYCLIC AMP (cAMP) which is produced from ATP via an enzyme is. The transport of glucose into the cell stimulates a signaling pathway that causes the instracellular concentration of cAMP to decrease because the pathway inhibits atp, the enzyme needed for cAMP synthesis. the effect of cAMP on the lac operon is mediated by an activator protein called CATABOLITE ACTIVATOR PROTEIN (CAP) - composed of two units, each of which binds one molecule of cAMP

***What is an operon?

a group of genes under the control of a single promoter

*** on its chromosome, an eocli cell is lac i negative, lac z, y, and a. It has an F' factor that is lac i,z,y,a positive. What is the expected level of expression of the lac operon genes (lac Z,Y,A) in the absence of lactose?

neither expressed

Why do operons occur in bacteria?

one biological advantage of an operon is that it allows a bacterium to coordinately regulate a group of genes that are involved with a common functional goal, the expression of the genes occurs as a single unit. for transcription to take place an operon is flanked by a PROMOTOR that signals the beginning of transcription and a TERMINATOR that specifies the end of transcription. Two or more genes are found between these two sequences.

conditions that favor the formation of the 3-4 stem loop

rely on the translation of the L gene. 3 scenarios are possible 1. Region 1 rapidly hydrogen bonds to region 2 and 3 is left to hydrogen bond to region 4 therefor the terminator stem loop forms and the transcription is terminated just past the L gene at the U-rich attenuator

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the ability of the lac repressor to bind to the operator site depends on whether or not allolactose is bound to it. Each of the repressor proteins four subunits has a single binding site for allolactose, the inducer.

lac operon has 3 operator sites for the lac repressor

O1 is the operator site slightly downstream from the promoter O2 is father down in the lacZ coding sequence. O3 upstream from the promoter If O2+3 are missing, repression is dramatically reduced even thwne O1 is present. When O1 is missing, even in the presence of the other operator sites, repression is abolished.

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a loss of function mutation in a gene encoding a repressor protein has the same effect as a mutation in an operator site that cannot bind a repressor protein. Both cases the genes of the lac operon are constitutivel expressed. in a merozygote the results are different. Whena normal lac i gene and a normal lac operon are introduced into a cell harboring a defective lac i gene, the normal lac i gene can regulate both operons. On contrast, a lac operon with a normal operator is introduced into a cell iwth a defective operator site, the operon coninues to be expressed without lactoe present.

*** Which of the following conditions would cause transcription to be activated?

a repressor plus an inducer

*** When an e coli is exposed to glu how does this affect the regulation of the lac operon via CAP?

cAMP does not bind to CAP and transcription is decreased

***A repressor is a _ that _ transcription

regulatory protein , inhibits

Fig 16.3

two units are present unit 1: lac operon contains a CAP site, promotoer (lacP), operator site (lacO), three structural genes (lacZ, Y, and A) and a terminator. lacZ encodes the enzyme beta-galactosidase - an enzyme that cleaves lactose into galactose and glucose. Also this converts a small perfecntage of lactose into allolactose (similar to sugar) and acts as a small effector molecule to regulate the lac operon lacY enodes lactose permease, a membrance protein required for the active transport of lactose into the cytoplasm

How does a small molecule like allolactose exert its effects?

when allolactose binds to the repressor, a conformational change occurs in the lac repressor protein that prevents it form binding to the operator site. Under these conditions, RNA polymerase is now free to transcribe the operon. In this process, it would be called being INDUCED - the action of a small effector molecule such as allolactose (ALLOSTERIC REGULATION)

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2.

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attenuation is a mechanism to regulat transcription that is found in several other operons involved with aa biosynthesis. the mrna that encode the leader peptides are rich in odons for the particular aa that is synthesizes by the enzymes encoded by the particular operon ie. the mrna that encodes the leader peptide histidine operon has seven hisitidine codons in its sequence and the mrna for the leader peptide of the leucine operon has 4 leucine codons.

Understanding how the induction process is shut off in a lactose-depleted environment

consider the interaction between allolactose and lac repressor The lac repressor has a measurable affinity for allolactose. the binding of allolactose to the lac repressor is reversible. The likeliood that allolactose will bind to the repressor depends on the allolactose concentration. During induction of the operon, the concentration of allolactose rises and approaches the affinity for the repressor protein. This makes it likely that allolactose will bind to the lac repressor thereby causing it to be released from the operator site.

the trp operon is regulated by a repressor protein

contains 6 genes trp L E D C B A EDCBA genes encode enzymes involved in tryptophan biosynthesis L plays a regulatory role R not a part of the trp operon encodes the TRP REPRESSOR protein. when levels within cell is low the trp repressor cannot bind to the operator site under this condition - RNA polymerase transcribes the trp operon. In this way the cell expresses the genes required fr the synthesis of tryptophan. when the tryptophan levels within the cell become high, tryptophan acts as a corepressor that binds to the trp repressor protein causing a conformational change in the trp repressor that allows it to bind to the trp operator site - this inhibits the ability of RNA polymerase to transcribe the operon therefor when a high level of tryptophan is present within the cell the cell doesnt need to make more - trp is then turned off

lac operon encodes proteins involved in lactose metabolism

in a bacteria it is common for a few genes to be arranged together in an OPERON - two or more genes under the transcriptional control of a single promotor operon encodes a POLYCISTRONIC RNA - contains the sequences of two or more genes

Rare mutation

in the lac-i gene that alter the regulation of the lac operon reveals that the lac repressor is composed of a protein domain that binds to the dna and another domain that contains the allolactose-binding site

Activator

regulatory protein that increases the rate of transcription. Positive Control

Inducer / INDUCIBLE GENES

small effector molecule that causes transcription to increase. An inducer can do this with two ways 1. bind to a repressor protein and prevent it from binding to the dna 2. bind to an activator protein and cause it to bind to the dna. Both cases - transcription rate s increased. Genes regulated from these two are called INDUCIBLE GENES

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terp opeorn downstream from the operator site is imporant during attenuation, the first gene in the trp operon is the L which encodes a peptide containing 14 a called the LEADER PEPTIDE. two features are key in the attenuation mechanism 1. twp tryptophan codons are found within the mrna that encodes the trp leader pepdide What is the role of the codons? these two codons provide a way to sense whether or not the caterium has sufficient tryptophan to synthesize its proteins 2. the mrna can form stem loops

the presence of a small effector molecule may INHIBIT transcription.

two ways 1. CORE REPRESSOR - a small molecule that binds to a repressor protein causing the protein to bind to the dna 2. INHIBITOR - binds to an activator protein and prevents it from binding to the dna both compressor/inhibitor act to REDUCE the rate of transcription there the genes they regulate are termed REPRESSIBLE GENES

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Now the lac repressor is unlikely to be bound to allolactose. When the allolactose is released the lac repressor returns to the conformation that binds to the operator site. The binding of the ressor shuts down the lac operon when lactose is depleted from the environment. mRNA and proteins encoded by the lac operon are eventually degraded.

Repressor

a regulatory protein that binds to the dna and INIBITS transcription. Negative Control

Regulation of the trp operon

encodes enzymes involved with the snthesis of the amino acid tryptophan. Regulated by a repressor protein and ATTENUATION - where transcription begins but is stopped prematurely.

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the effect of glu called CATABOLITE REPRESSION involves the action of an inducer cAMP and an activator protein CAP, not a repressor.

lac-i gene encodes a diffusible repressor protein

Paradee identitfied a few rare mutant strains of bacteria that had abnormal lactose adaptation. lac-i negative resulted in the constitutive expression of the lac operon even in the abscence of lactose.

Observations concluded that

enzyme adaptation is due to the synthesis of specific cellular proteins in response to lactose in the environment.

Allosteric poteins

have at least two binding sites, the effector molecule binds to the proteins ALLOSTERIC SITE, which is a site other than the proteins active site.

Unregulated Genes/ Constitutive Genes

have constant levels of expression in all conditions over time. Encode proteins needed for survival.

the regulation of the lac operon allows bacterium to respond to environmental change

in the absence of lactose, no inducer is available to bind to the lac repressor. The lac repressor binds to the operator site and inhibits transcription. When bacterium is exposed to the lactose (a very small amount) can be transported into the cytoplasm via lactose permease and beta galactosidase converts some of it to allolactose.

lac operon is also regulated by an activator protein

lac operon can also be transcriptionally regulated in a second way, CATABOLITE REPRESSION. This is influenced by the presence of glucose which is a catabolite - a substance that is broken down inside the cell the presence of flucose leads to repression of the lac operon. When exposed to both glucose and lactose, ecoli cells use glucose and catabolite repression prevents te use of lactose. Why is this an advantage? bacterium does not have to express all of the genes for both glu and lac metabolism. if the glucose is used up, catabolite repression is alleviated and the bacterium expresses the lac operon.

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production of merozygotes was instrumental in allowing Paradee to elucidate the function of the lac-i gene 2 key points 1. two lac-i genes in a merozygote may be different alleles ie. lac i gene on the chromosome may be lac i negative allele that causes constitutive expression whereas the lac i gene on the F' factor may be normal 2. the genes on the F' factor and the genes on the bacterial chromosome are not physically adjacent to each other.

Small Effector molecules

do not bind directly to dna to alter transcription - Effectors exert its effects by binding to an activator or repressor. The binding of the effector molecule causes a conformational change in the regulatory protein influencing whether or not the protein can bind to dna. Genetic regulatory proteins that respond have two functional domains 1. A site where the protein binds to the dna 2. the binding site for the effector molecule

Processes regulated at the genetic level

Metabolism- some proteins function in the metabolism of small molecules. For example, some enzymes are needed for a bacterium to metabolize certain sugars. Response to environmental factors- Some proteins help a bacterium to survive environmental stress like osmotic/heat shock. Cell Division-some proteins are needed for cell division.

Translational and Post translational regulation

Post translational regulation refers to the functional control of proteins that are already present in the cell rather than regulation of transcription or translation. Can either activate or inibit the function of a protein. Compared with transcription control can be fast (an advantage) transcriptional and translational regulation typically require several min or even hours to take effect because tow mechanisms involve the synthesis and turnover of mrna and polypeptides.

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Region 2 is complementary to 1 and 3. Region 3 is complementary to 2 and 4. The three stem loops are possible 12, 23, 34 ie. if region 2 forms a stem loop with region 1 it cannot at the same time form a stemloop with region 3. then region 3 cannot form a stemloop with region 4. 34 stemloop is fuctionally unique. they stemloop with the U-rich attentuator for sequence acts as an intrinsic terminator a P-INDEPENDENT terminator. the formation of 34 stemloop causes rna polymerase to pause and the U-rich seq to disassociate from the dna. this terminates transcription at the U rich attenuator

transcriptional regulation

at the rate of RNA synthesis can be increased or decrease, not necessarily turned off or on. involved the actions of regulatory proteins that can bind to the dna and affect the rate of transcription of one or more nearby genes. 2 Types of regulatory Proteins - 1 Repressor 2 Activator

trp operon is also regulated by Attenuation

can occur in bacteria because the processes of transcription and translation are coupled. Bacterial ribosomes quickly attach to the 5' end of the mRNa soon after its synthesis begins via RNA polymerase. During attenuation, transcription actually begins but is terminated before the entire mRNA is made. A segment of dna termed ATTNUATOR SEQUENCE is important in facilitating this termination. When attenuation occurs the mRNA from the trp operon is made as a short piece that terminates at the attenuators sequence which is shortly past the trpL gene. because this short mrna has be terminated before rna polymerase has transcribed EDCBA genes it does not encode the protein required for tryptophan biosynthesis. attenuation inhibits the further production of tryptophan in cell

CAP

determines whether the lac operon is expressed in the presence or absence of lactose/glu. when lac is only present, cAMO levels are high. cAMP bind to CAP and then CAP binds to the CAP site and stimulates the ability of rna polymerase to begin transcription. In the presence of lactose, the lac repressor is not bound to the operator site so transcription can proceed at a high rate. Abense of both lac and glu, cAMP are also high, but the binding of the lac repressor inhibits transcription even though CAP is bound to the dna. Therefor transcription rate is very low

repressor proteins and antisense rna can inhibit translation

for some bacterial genes, mrna is regulated by the binding of proteins that influence the ability of ribosomes to translate the mrna into polypeptide. TRANSLATIONAL REGULATORY PROTEIN recognizes a seq within the mrna. TRANSLATIONAL REPRESSORS - proteins act to inhibit translation. When binded to an mrna, it can inhibit by 2 ways 1. can bind in the vicinity of the shine dalagarno seq and or the start codon thereby blocking the ribosomes ability to initiate translation in the region. the repressor protein may bind outside the shine dalagarno/start codon region but stabilize an mrna secondary structure that prevents initiation.

The phenomenon of enzyme adaptation is due to the synthesis of cellular proteins

francois Jacob and Jacques Monod Enzyme Adaptation - the observation that a particular enzyme appears within a living cell only after the cell has been exposed to the substrate for that enzyme. When a bacterium is not exposed to a particular substance it does not make the enzymes needed to metabolize that substance.

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lac-i negative mutations that result in the contitutive expression of the lac operon aka it is expressed in teh presence and absence of lactose this may result in an inability to synthesize any repressor protein or they may produce a repressor protein that is unable to bind to the DNA at the lac operator site. if the lac repressor is unable to bind to the DNA, the lac operon cannot be repressed

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lacA encodes galactoside transacetylase, an enzyme that covalently modifies lactose and lactose analogs.

Bacterial Conjugation

mattings between recipient cells termed F- and donor cells (Hfr) transferred a portion of the bacterial chromosome. Sometimes an F factor also carries genes that were originally found within bacterial chromosome. These types of F factors are called F' factors. In their studies, identified F' factors that carried the lac-i gene and the lac operon. These F' factors can be transferred form one cell to another by bacterial conjugation. A strain of bacteria containing F' factor genes is called a MEROZYGOTE/ PARTIAL DIPLOID

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the cap site and operator site are short dna segments that function in gene regulation. CAP SITE - a dna sequence recognized by an activator protein called the catabolite activator protein OPERATOR SITE - a sequence of bases that provide a binding site for a repressor protein

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the cytoplasmic level of allolactose gradually rises and allolactose binds to the lac repressor. The binding of allolactose promites a conformational change that prevents the repressor from binding to the lac operator site therefor allowing transcription of the lac Z,Y, and A genes to occur. Translation of the encoded polypeptide produces the proteins needed for lactose uptake and metabolism.

lac-iS mutations have the opposite effect

the lac operon cannot be induced even in the presence of lactose. These mutations are typically located in the domain that binds allolactose. The mutation usually results in a lac repressor protein that cannot bind the allolactose. If the lac repressor is unable to bind allolactose, it will remain bound to the lac operator site and therefore induction cannot occur.

Interaction between regulatory protein and dna sequences illustrated in this experiment have leg to the definition of two genetic terms.

TRANS EFFECT - a form of genetic regulation that can occur even though two dna segmenta are not physically adjacent. the action of the lac repressor on the lac operon is a trans effect TRANS ACTING FACTOR - a regulatory protein like the lac repressor CIS ACTING ELEMENT - is a dna segment that must be adjacent to the genes that it regulates and it is said to have a CIS EFFECT on gene expression. ie. lac operator site = cis acting element trans effect is mediated by genes that encode regulatory proteins whereas a cis effect is mediated by dna sequences that are bound by regulatory proteins.

16.9

Repressor must bind to O1,2,3to cause full repression. The lac repressor can bind to O1+2 or O1+3. Never O2+3. If either O2 or O3 were missing, maximal repression is not acheived because it is less likely for the repressor to bind when only two operator sites are pretend binding of the lac repressor or two operator sites requires the dna to form a loop which would bring the operator sites closer together the binding of cAMP-CAP complex to the CAP site (blue protein within loop) causes a 90 degree bend in the dna structure. When the repressor is active, not bound to allolactose, the cAMO-CAP complex facilitates the binding of the lac repressor to the O1+3 site.