Chap 16

Negative control:

(feedback) A negative feedback loop is a reaction that causes a decrease in function. It occurs in response to some kind of stimulus. Often, it causes the output of a system to be lessened; so, the feedback tends to stabilize the system.

Positive Control:

(feedback) Positive feedback is a process in which the end products of an action cause more of that action to occur in a feedback loop. This amplifies the original action. It is contrasted with negative feedback, which is when the end results of an action inhibit that action from continuing to occur.

List all of the parts of the lac and trp operons and discuss the function of each part. This should prepare you to answer questions about the trp and the lac operons. eg. Explain how glucose levels are assessed by the lac operon. eg. Describe what will happen to the trp operon if tryptophan is low.

Lac operon : Promoter (P): promoter for I gene. Required for transcription initiation of the operon. Also another promoter for z y a I = gene product binds operator : Encodes Lac repressor O = operator = repressor binding site Structural genes : Lac Z gene : Encodes β galactosidase which cleaves lactose into glucose and galactose by breaking the β 1-4 linkage. It also cleaves allolactose into glucose and galactose by breaking the β 1-6 linkage. Lac Y gene : Encodes permease which transports lactose into bacterial cell. Lac A gene : Encodes transacetylase which detoxifies compounds having acetyl group. No role in lactose metabolism. Lac operon process • In the absence of lactose (inducer), Lac repressor is produced from the promoter which binds to the operator region, thus shutting down the operon. This is known as negative regulation. • In the presence of lactose, basal level of β galactosidase present in the cell cleaves the lactose into allolactose. Alloloctose which is the actual inducer, binds to the repressor rendering it inactive. Thus, the repressor can no longer bind to the operator. All the genes are structural genes are transcribed. • Bacterial cell uses lactose only when the glucose levels are low. Glucose levels are assessed by the concentration of cAMP (cyclic AMP). Concentration of cAMP and concentration of glucose are inversely related. • CRP (cAMP receptor protein) or CAP (Catabolite activator protein) is a positive regulator of lac operon. In the absence of glucose, the intra-cellular cAMP levels rise. This activates the CAP which binds to its site (upstream of the lac promoter) and contacts the RNA polymerase at the promoter, thereby activating transcription. Trp operon : Trp R : encodes an inactive repressor called apo-repressor. Trp P : Promoter which is required for transcription initiation of the operon Trp O : Operator where repressor binds. Trp L : Encodes a leader peptide which is involved in attenuation. Trp E, Trp D, Trp C, Trp B, Trp A : Encodes proteins which are required for the biosynthetic pathway of tryptophan. Trp operon regulation : Repression • In the absence of tryptophan, apo-repressor is formed by trp R which remains in an inactive form and cannot bind to the operator. The operon is switched on. • In the presence of tryptophan, tryptophan acts like a co-repressor that binds to the apo-repressor making it a fully active repressor. This active repressor binds to the operator and turns off the operon. Attenuation This mechanism is mediated by the transcripts produced from the leader peptide. The mRNA transcript of leader peptide consists of four regions which can base pair to form secondary structures. Base pairing of region 1 and 2 : pause signal Base pairing of region 3 and 4 : termination of transcription signal Base pairing of region 2 and 3 : anti-termination signal Crucial to attenuation is the tight coupling of transcription and translation. Region 1 and 2 base pair after they have been synthesized by RNA polymerase. This pauses the RNA polymerase so that ribosome can load onto the mRNA. When tryptophan levels are low, very few are available for amino acylation of the tRNA. Charged trp-tRNA levels drop and the ribosome stalls at region 1. This enables the region 2 to base pair with region 3 forming an anti-termination signal. The RNA polymerase continues to synthesize the structural genes. When tryptophan levels are high, ribosome continues past the region 1 to region 2 (due to the availability of charged Trp-tRNA). Since the ribosome covers region 2, it cannot base pair with region 3. Region 3 base pairs with region 4 forming the termination signal.

Discuss how Riboswitches can regulate gene expression.

Through changes in RNA secondary structure. Regulation is achieved by altering the secondary structure of RNA through binding of a small effector molecule (ligand) to the RNA (aptamer = a RNA domain that binds a ligand). This mechanism is named riboswitch regulation because the small molecule directly binds to the mRNA causing a 'switch' in the secondary structure of mRNA. Riboswitches regulate expression of genes only at the level of transcription or only at translation. Riboswitches are typically found downstream of the genes they regulate.

Operator:

a genetic sequence which allows proteins responsible for transcription to attach to the DNA sequence. The gene, or genes, which get transcribed when the operator is bound are known as the operon. ... The function of the operator within genetics is to regulate the production of a certain portion of the DNA.

Inducer:

a molecule that regulates gene expression. An inducer functions in two ways; namely: By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator.

Promoter:

a promoter is a sequence of DNA to which proteins bind that initiate transcription of a single RNA from the DNA downstream of it. This RNA may encode a protein, or can have a function in and of itself, such as tRNA, mRNA, or rRNA.

Attenuation:

a regulatory mechanism used in bacterial operons to ensure proper transcription and translation. In bacteria, transcription and translation are capable of proceeding simultaneously.The process of attenuation involves the presence of a stop signal that indicates premature termination.

Repressor:

a repressor is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA.

cAMP:

a second messenger important in many biological processes. cAMP is a derivative of adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway.

Corepressor:

a substance that inhibits the expression of genes. In prokaryotes, corepressors are small molecules whereas in eukaryotes, corepressors are proteins.

Repressible operon:

operon that are on by default but can be turned off by a small molecule.

Riboswitches:

specific components of an mRNA molecule that regulates gene expression. The riboswitch is a part of an mRNA molecule that can bind and target small target molecules. ... Riboswitches are found in bacteria, plants, and certain types of fungi.

Antiterminator:

the prokaryotic cell's aid to fix premature termination of RNA synthesis during the transcription of RNA. It occurs when the RNA polymerase ignores the termination signal and continues elongating its transcript until a second signal is reached.

Regulator gene:

A regulator gene, regulator, or regulatory gene is a gene involved in controlling the expression of one or more other genes. ... In prokaryotes, regulator genes often code for repressor proteins. Repressor proteins bind to operators or promoters, preventing RNA polymerase from transcribing RNA.

Operon:

A set of genes transcribed under the control of an operator gene. More specifically, an operon is a segment of DNA containing adjacent genes including structural genes, an operator gene, and a regulatory gene. An operon is thus a functional unit of transcription and genetic regulation.

Structural genes:

Any of the genes coding for the production of a specific RNA, structural protein, or enzyme not involved in regulation. Supplement. In an Operon Model, structural genes are those genes that control the production of a protein that performs a structural role in cell.

Inducible operon:

Each operon contains regulatory DNA sequences, which act as binding sites for regulatory proteins that promote or inhibit transcription. Some operons are inducible, meaning that they can be turned on by the presence of a particular small molecule.

Explain the regulation of the trp operon by the 5' end of the mRNA when tryptophan is low vs high.

The 5' end of the mRNA of trp operon consists of: Regulatory region (mRNA for repressor protein) and Sructural region (protein components of trp E/D/C/B/A). High trp- trp will bind with this repressor protein and attach to the operator site. This will prevent RNA pol from moving over DNA, no mRNA formed and no protein. Low trp- DNA at 3' end transcribe into 5' end of mRNA and form repressor protein. Since trp is less, repressor is inactive and doesn't bind to operator. Tryptophan forming proteins will be formed.

CAP:

a DNA binding protein involved with the transcription of several genes, including those that code for enzymes involved in the metabolism of certain sugars (i.e. lactose, maltose, and arabinose.)

DNA binding proteins:

proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because it exposes more functional groups that identify a base pair.