Molecular Biology Unit 8

List several differences between eukaryotic and prokaryotic transcription

1) Eukaryotic mRNA contain a 5' cap and poly-A tail whereas prokaryotic mRNA have neither 2) Eukaryotes have 3 RNA polymerases. RNA Pol II makes most mRNA's (it is the main one). Prokaryotes have 1 RNA pol II 3) Eukaryotes require many general transcription factors. These are proteins that assemble at the promoter to help initiate transcription. Prokaryotes only require a sigma factor 4) Repressors and Activators may bind to DNA sequences that are thousands of bp away. 5) Eukaryotic transcription must deal with the packaging of DNA into nucleosomes. Prokaryotes do not.

Eukaryotic txn: What are the 3 ways by which eukaryotic gene expression is controlled?

1) activators/repressors (transcription regulators) 2) by coordinating expression of multiple different genes within a single protein (one activator can activate many genes) 3) by regulating chromatin state around proteins (not a factor for eukaryotic cells)

How do repressors function:

1) binding to DNA sites that overlap with activator binding sites (compete for the same region of DNA) 2) through protein-protein interactions

How can gene expression be regulated? (CT, CS, S--> N to C, D, S--> P by R, PD)

1) controlling when/how often a given gene is transcribed 2) controlling how an RNA transcript is spliced/processed 3) selecting which mRNAs are exported from the nucleus to the cytosol 4) mRNA degradation 5) selecting which mRNAs are translated into protein by ribosomes 6) protein degradation

Eukaryotic txn: What are some reasons spacing of promoter elements could be important?

1) movement of the DNA element to the other face of the helix could constrain the regulatory proteins (activators/repressors) from interacting with the basal transcription machinery --> preventing gene expression 2) modifying the distance between transcription factor binding elements could impact the ability of transcription factors from activating transcription

General transcription factors help the...

1) polymerase molecule to correctly bind, or align properly at the promoter 2) pull apart 2 strands of DNA 3) allow RNA polymerase to leave the promoter as transcription begins

How do cells create cell memory to transmit them to their daughter cells?

1) positive feedback loops 2) chromatin structure 3) DNA methylation

For the activation of eve, what are the activators and what are the repressors?

Bicoid and Hunchback activate and Kruppel and Giant repress

Lac Operon in E.Coli: What is the Lac operon controlled by?

By both an activator and a repressor. Transcription of the lac operon is controlled by the availability of both glucose and lactose

Lac Operon in E.Coli: If glucose is present then the lac operon is turned...

OFF

Lac Operon in E.Coli: If glucose is absent and lactose is present then the lac operon is turned...

ON

General Transcription Factors (ex: TFIID, TFIIB)

Proteins that assemble at the promoter to help initiate transcription. Needed and used at all eukaryotic promoters

Transcription regulators/Gene regulatory proteins

Proteins that binds to regulatory DNA sequences and control whether a gene is switched on or off

What is the primary function of gene control in eukaryotes?

control precise developmental decisions

What is DNA methylation associated with?

decreased gene expression because it blocks transcription

How is cell type maintained?

differential expression and cell memory

The frog experiment demonstrated that DNA is not irreversibly removed during...

differentiation

How do cells retain memory of their chromatin state?

each daughter chromosome will inherit about half of its parent's collection of modified histones (thus they have a memory of their chromatin structure)

Eukaryotic txn: The DNA sites to which eukaryotic gene activators bind are termed

enhancers

Eukaryotic txn: How does the arrangement of chromosomes into looped domains keep enhancers in check?

ensures each gene is associated with the proper enhancer

What type of signals trigger differential expression of proteins? (E,CC,MG,SM)

environment, cell contacts, molecular gradients, signaling molecules

Differential Expression of Proteins: Specialized cells are capable of altering their patterns of gene expression in response to...

extracellular signals and developmental programs

Differential expression

genes allow specific proteins to be expressed in different cells

Eukaryotic txn: Unlike prokaryotic regulatory sequences, eukaryotic regulatory sequences act over

greater distances (The transcription of a eukaryotic gene can be influenced by additional gene regulator proteins that bind near and far from the promoter)

Eukaryotic txn: What do repressors attract to reverse the positive effects that histone-modifying enzymes have on transcription initiation?

histone deacetylases

Eukaryotic txn: Why do eukaryotic transcriptional activators recruit histone-modifying enzymes?

histone-modifying enzymes serve as binding sites for proteins to stimulate transcription

Common proteins that are expressed in all cells are referred to as...

housekeeping genes

When a cell divides by mitosis, it produces two daughter cells with...

identical DNA content (patterns of gene expression can be transmitted to daughter cells to maintain their identity)

Bacteria often coordinate the expression of a set of genes by having them clustered together

in an operon under the control of a single promoter (in eukaryotes gene transcription is regulated individually)

What are the purpose of master regulators?

induce cells to differentiate into other cell types and create combinations of transcription factors that can be used for cell memory

Lac Operon in E.Coli: In the presence of both glucose and lactose, what state is the lac operon in?

it is turned off

Lac Operon in E.Coli: Cells utilize glucose as an energy source first; however, if there is no glucose available what do cells use?

lactose

Eukaryotic txn: How does DNA looping in eukaryotes allow for gene activation to occur at a distance?

looping of DNA allows for contact between the activator and the transcription initiation complex bound to the promoter

Eukaryotic txn: What does histone acetylation by histone acetyltransferase do?

loosens the hold on DNA improving access for DNA regulator proteins

Lac Operon in E.Coli: In the absence of glucose and the presence of lactose, what occurs?

low glucose levels activates cAMP --> cAMP binds to the CAP activator --> complex binds to DNA promoter to activate transcription of lac operon genes

What can a combination of only a few transcription regulators generate?

many different cell types that can be used for memory (positive feedback loop)

Cytoplasmic determinants

maternal substances in the egg that influence early development (mRNA & proteins)

Polycistronic

multiple genes encoded by the same promoter region (occurs in prokaryotes)

Do eukaryotes contain operons?

no, gene transcription is regulated individually

Prokaryotic example - Tryptophan Synthesis: When can the Trp repressor protein bind to DNA?

only when it is bound by tryptophan

How can the expression of a gene such as eve in stripe 2 be regulated to only be expressed in stripe 2?

overlapping repressor and activator sites in the promoter of stripe 2 only activates expression of Eve in the presence of activators and the absence of repressors

How do daughter cells remember what kind of cells they are supposed to be?

positive feedback loops (a master transcription regulator activates transcription of its own gene, in addition to that of other cell-type- specific genes. Each time a cell divides, the regulator is distributed to both daughter cells, where it continues to stimulate the positive feedback loop)

Signals cause transcriptional regulators to bind to regulatory DNA sequence elements in order to...

switch genes on and off (they act as activators and repressors)

Eukaryotic txn: Coordinating expression of multiple different genes within a single protein requires...

that each promoter has the DNA element for the regulator

How is tissue specific gene expression controlled?

the arrangement of promoter elements and local expression of transcription regulators (no single arrangement of sites dictates tissue specific expressions/need different arrangement in each tissue type

What is gene expression determined by?

the combination of the DNA elements within the promoter and the presence/absence of transcription factors that bind them

How does an individual cell specify which of its genes to express?

via differentiated cells --> cell types contain the same genes but express them differently

Lac Operon in E.Coli: When is the lac operon expressed?

when glucose is absent and lactose is present --> CAP activator binds to the promoter and the repressor is off (lactose binding to the repressor removes the repressor from the DNA to allow the activator to turn on transcription in the absence of glucose)

Prokaryotic example - Tryptophan Synthesis: When do we want to tryptophan operon off?

when tryptophan is high --> trp bind to the repressor to keep the genes that would make trp off

Prokaryotic example - Tryptophan Synthesis: When do we want to tryptophan operon on?

when tryptophan is low --> RNA polymerase binds to the promoter and transcribes the genes

What does combinatorial control allow for?

cell type specific control

How are cytoplasmic determinants distributed in an unfertilized egg?

unevenly in different regions which leads to different gene expression

Eukaryotic txn: Why do eukaryotic transcriptional activators recruit chromatin-remodeling complexes?

chromatin-remodeling complexes make DNA packaged in nucleosomes more accessible to proteins by exposing the TATA box

What are eukaryotic genes regulated by?

combination of proteins (combinatorial control)

What is the main difference between the tryptophan repressor and the lac repressor?

- when trp levels are high the trp repressor binds to prevent transcription - when lactose is preset the lac repressor is switched off to initiate transcription in the absence of glucose

What does an operon consist of?

A promoter, an operator, and one or more functional genes

Gene expression

Cells selectively direct the synthesis of thousands of proteins and RNAs encoded in their genome

The process by which groups of transcription regulators work together to determine the expression of a single gene

combinatorial control

Eukaryotic txn: What are distal enhancers?

DNA elements that are recognized by regulatory transcription factor proteins (enhancer/activator, TATA box) (there is sequence specificity so that the protein will only bind to specific DNA sequence)

Aside from positive feedback loops and memory of their chromatin state, what is an additional mechanism used to reinforce cell identity?

DNA methylation

This covalent modification generally turns off the affected genes by attracting proteins that bind to methylated cytosines and block gene transcription Prior to DNA replication, methylated cytosines are on both the template and non-template strands --> after DNA replication only the template strand is methylated --> proteins recognize methylation and add it to the newly synthesized daughter strand

DNA methylation

In order to fit in the major or minor groove, there are common structures that the polypeptides will need to take on. What are these referred to as?

DNA-binding motifs

What provided proof that differentiated cells contain all the genetic instructions to form a complete organism? (Experiment: The nucleus is taken from a skin cell in an adult frog and injected into a frog egg from which the nucleus has been removed.)

Implantation of a nucleus from a differentiated cell (such as a frog skin cell) into an enucleated egg resulted in the development of an entire embryo. To make these different cell types, the nucleus of the differentiated cell must have had all the genetic information for the entire organism. (the doctored egg developed into a normal tadpole meaning that the nucleus from the transplanted skin cell did not lose any critical DNA sequences which tells us the nucleus has all the information needed to create an organism)

Lac Operon in E.Coli: What occurs when lactose is absent

Lac repressor binds to the Lac operator and shuts off expression of the operon (opposite of tryptophan --> when trp is absent they want more to be made)

Prokaryotic example - Tryptophan Synthesis: When the operator is bound by the Trp repressor protein what occurs?

RNA polymerase cannot bind to the promoter

What are examples of housekeeping genes?

RNA polymerases, DNA repair enzymes, ribosomal proteins, TFIID

T/F: The transcription regulators and the locations of their DNA binding sites relative to the promoters are different for different genes.

T

Explain how myoD creates a positive feedback loop which helps create cell memory:

a transient signal leads to the expression of myoD --> initial expression of the myoD transcription factor keeps the transcription of the myoD gene always on

Operon

a series of adjacent genes that are all transcribed into a single mRNA --> polycistronic only occurs in prokaryotes

What can coordinate the expression of different genes?

a single protein (in the absence of the glucocorticoid hormone GR protein receptor is blocked and genes are expressed at a low level --> in presence of glucocorticoid hormone genes are expressed at high levels)

An ________________ _________________ binds to a ________________ _________________ on the DNA and then interacts with the RNA polymerase to help initiate transcription

activator protein; regulatory sequence

Cells have the ability to change which genes they express WITHOUT

altering the nucleotide sequence of their DNA

Eukaryotic txn: In a eukaryotic cell, what does the promoter contain?

basal machinery elements (initiation site and sequences required for pol II to bind - TATA) and regulatory sequences

Why are repressors referred to as allosteric proteins?

because they have two or more conformations

Lac Operon in E.Coli: How is the lac operon positively regulated?

by cAMP

Differential expression of transcriptional regulators leads to...

cell diversity/contributes to cell memory

The memory of previous transient signals

cell memory

Eukaryotic txn: How do transcriptional activators influence chromatin structure?

recruit histone-modifying enzymes and chromatin remodeling complexes that impact the condensation of the DNA packaging

How is the expression of Eve in the other strips controlled?

regulated by other regions of the promoter that have a different arrangement of promoter elements --> many of the DNA elements are bound by other proteins that do not impact stripe 2 expression.

Eukaryotic txn: Although the mediator complex does not touch DNA directly, what does it interact with?

regulatory DNA sequences and general transcription factors

What are some examples of non-housekeeping genes?

regulatory factors or special splice variants

Within the operon's promoter is a short DNA sequence, called the operator, what is the operator recognized by?

regulatory proteins - activators and repressors

The binding of regulators to DNA (activators and repressors) are regulated by the interaction of what?

signaling molecules (regulate conformational changes in 3-D structure so that the protein can bind to the operator sequence)

What is the function of the operator in prokaryotes?

sits between the -10 and -35 promoter sites; a regulatory DNA element that serves as a repressor binding site

Describe combinatorial control

small numbers of transcription factors can control the expression of a much larger number of genes (Multiple elements usually control a gene, so many combinations are possible --> it allows for cell type specific control)

Eukaryotic txn: What does the DNA-binding domain interact with? What does the activation domain interact with?

specific DNA sequences; other proteins to stimulate transcription

Promoter

specific region of a gene where RNA polymerase can bind and begin transcription

Eukaryotic txn: When repressors/activators bind to the distal elements of the DNA loop the double helix _____________ ________________ and hydrogen bonds _________________ __________________

stays intact; aren't broken

Prokaryotic example - Tryptophan Synthesis: Low amounts of repressor protein are always made to ensure that the repressor protein is around so expression regulation depends on what?

the levels of tryptophan

Within the DNA sequence, what does the repressor bind to?

the operator; a small repressor sequence within the promoter region

Prokaryotic example - Tryptophan Synthesis: What is the tyrptophan (trp) repressor regulated by?

the presence or absence of tryptophan

How do positive feedback loops work?

the product causes more product to be made by keeping transcription on for a particular protein (MyoD regulates its own transcription and has binding sites in the promoter region of several other genes that are needed to make muscle specific proteins --> tells progeny to make muscle proteins)

Different cell types of a multicellular organism contain...

the same DNA/genes but express them differently

What is the primary function of gene control in prokaryotes?

to adjust to its immediate nutritional and physical environment

Eukaryotic txn: What influences or prevents assembly of the basal transcription initiation complex or attract proteins that modulate chromatin structure?

transcription factors (either causing condensation or allowing access to the DNA regions required, depending on the type of modulator)

What can be used to experimentally direct the formation of specific cell types in culture?

transcription regulators

What must occur in order to switch genes on and off?

transcriptional regulators bind to a specific nucleotide sequence in the major groove of the double helix