Gene Regulation—Prokaryotes

What are the three components of a regulatory switch. Give some specific examples

1. Regulatory seq (element) that is cis acting= operator seq= which detect regulation. 2. Regulatory protein that is trans acting (lac I repressor) 3. Signal molecule that is trans acting. Signals condition in the cell (lactose). Must have all three components for regulation

What is the difference between always off and always on mutants? Which would you be more interested in studying and why?

Always off mutants: No gene expression. Mutation may be in regulatory sequence or the gene sequence itself Always on mutants: Regulation is affected, not the gene sequence. These are the most interesting for studying purposes because you can do complementation experiments to restore wild type functionality and thus learn about the regulatory mechanisms

Define operon and regulon

An operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. A regulon is a collection of genes or operons under regulation by the same regulatory protein. This term is generally used for prokaryotic systems and is related to combinatorial control

What is combinatorial control? Give an example

Combinatorial control is when different operons have regulatory sequences and regulatory proteins in common, so that they can be regulated by the same signals. The sugar utilization regulon is an example of combinatorial control.

gene regulation - prokaryotes. general idea

Gene regulation: Operons. Operons a group of genes that are co-regualted. An operon consist of a promoter seq. and a operator seq. followed by several related genes. An example of gene regulation in prokaryotes is the Lactose Operon. The lactose operon is the regulation of how the cell acts in the presence of lactose. Jacob and Munod were the ones to introduce gene regulation. They found that mutants are needed to study gene regulation. The Lac Operon has the following components: Lac I, Cap site, Promoter + operator, lac z , lac y and lac a. The lac I which is upstream from the the primer/operon which produces a repressor that acts on the operator site the lac I gene has its own promoter. The promoter binds the RNA polymerase. The Operator binds with the repressor , not really bind but interacts with the repressor. The Lac z gene codes for b-galactosidase which cleaves lactose in glucose and galactose. The Lacy y gene codes for permease. Lac A codes for transctase. In the presence of lactose. Lactose will bind to the repressor to prevent it to bind/interact with the operator. If the operator is unbound=promoter binds RNA polymerase. transcription of the gene occurs. Lac z,y and a is always constantly transcrived at low levels. If lactose is not present at all= the Cap protein will become activated it can induce transcription at a very low rate.

Explain how the lactose operon repressor is a negative inducible system.

In negative inducible operons, a regulatory repressor protein is normally bound to the operator and it prevents the transcription of the genes on the operon. If an inducer molecule is present, it binds to the repressor and changes its conformation so that it is unable to bind to the operator. This allows for expression of the operon....the operon needs to induce to do transcription. Lac I gene produces the repressor...in the presence of lactose, lactose is converted into allolactose which would bind to the repressor so the repressor can't bind to the operator. This allows the promoter to recruit RNA polymerase and transcription occurs. It is important to note that even without an inducer, a small amount of transcription still occurs due to the binding equilibrium of the repressor. This is important for the lac operon's function.

define Promoters

Location where RNA polymeraae bind to DNA before initiating transcription. Are cis-acting control meaning that the have to be on the same side of where DNA polymerase should be binding.

What is the difference between negative and positive regulation?

Negative regulation: a repressor binds to operator to prevent transcriction Positive regulation: an activator protein stimulates transcription by binding to DNA (cap protein)

define Repressor

Proteins that interfere with the transcription of their target gene. They can be find close to or at a distant from the operon they are repressing (trans acting). Repressors are usually made at a constant rate. In bacteria repressors interfere directly with RNA polymerase binding to a gene.

What subunit of prokaryotic RNA polymerase is necessary to initiate transcription? What does it interact with?

RNA polymerase in prokaryotes have alpha, beta, and sigma subunits. The sigma factor is the subunit that directs general transcription activities. E.g. s70 is the normal one, sHS causes cells to transcribe different genes to react to stressors, and sSPO directs RNA pol to sporylation. The sigma factor interacts with the core of the RNA polymerase, presumably altering its structure

define Inducible

Refers to the ability of a protein to be positively induced by a signal molecule

define Repressible

Refers to the ability of a protein to be repressed by a signal molecule

What is signal integration? Give an example.

Signal integration is when multiple regulators of a gene or operon are present. An example is the lac operon, which has signals from lactose and cAMP. These signals interact with different proteins, affecting transcription rates.

How does the CAP protein regulate transcription of the lactose operon? Is this positive or negative regulation?

The CAP site/protein provides another layer of regulation for when both glucose and lactose are present. When both are present, the repressor cannot bind (because lactose blocks it), but transcription is still low. This is because RNA pol needs to be activated before quick transcription can occur. In the lac operon, it is activated by the CAP protein - cAMP complex. cAMP is only produced when glucose levels are low. So when glucose is low, cAMP binds to CAP, which sticks to the CAP site and interacts with the nearby RNA pol to activate it (they interact through the CTP: carboxyl terminal domain).

Describe how a complementation experiment works. What is the difference between trans and cis complementation?

The complementation experiment works by adding elements to mutants to try to figure out how regulation works. If you have mutation in the promoter, no transcription occurs and no transcription occurs if you add an active promoter to the cell (promoter is cis acting). An operator is also cis acting. A mutant repressor causes transcription to occur. If you introduce a working repressor to the cell, you can turn the gene off. Thus, repressors are trans acting (i.e. the repressor can diffuse through the cell and bind to the DNA)

Explain the factors that determine whether λ phage undergoes the lytic or lysogenic cycle. How does a lysogenic phage get converted into a lytic phage?

When a bacteria is infected, phage repressor keeps the cell in lysogenic cycle. The phage repressor is a dimer that binds to the phage operator sequence, blocking transcription. When bacterial DNA is damanged, it produces a DNA damage signaling protease, which cleaves the phage repressor in half. This causes transcription to begin, and the phage enters the lytic cycle, eventually causing the host to lyse.

define Co-Repressor

a co-repressor is a substance that inhibits the expression of genes. A co-repressor represses the expression of genes not through direct interaction with a gene promoter (a sequence of DNA adjacent to the regulated gene), but rather indirectly through interaction with repressor proteins that in turn bind to the promoter. In prokaryotes, the term co-repressor is used to denote the activating ligand of a repressor protein. For example, the E. coli tryptophan repressor (TrpR) is only able to bind to DNA and repress transcription of the Trp operon when its co-repressor tryptophan is bound to it. In eukaryotes, a co-repressor is a protein that binds to transcription factors

define Operon

a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter

define Operator

gene region that has an infinity with the repressor. Regaltory seq. kinda tells when transcription can occur

difference between positive/negative regulation and inducible/repression

positive/negative regulation refers to the effect on transcription when the regulatory protein is bound to the regulatory sequence. Inducible/repressible refers to the effect of the signal molecule

What is a consensus sequence? How do they relate to promoters?

promoter strength depends on resemblance to consensus sequence A consensus sequence determines where RNA polymerase will bind to. The consensus sequence is defined as a sequence that is complementary to the RNA pol binding site. The closer the promoter is to the consensus sequence, the stronger it will be

define Co-activator

same as co-repressor, opposite effect

define Inducer

signal molecule that starts gene expression by disabling repressor proteins. Allolactose is an inducer