Review and Summary

 

BIOC/MCB 568 -- Fall 2010
John W. Little--University of Arizona

BIOC/MCB568 Home Page 

 

NOTE: The following summarizes most of the important points for Little's section of the course bearing on transcription and its regulation. It was prepared in 2000, so some new material has not yet been included. Nonetheless, it will serve as a general guide to what I think is important. Also, be aware that if I talk about a topic in the lectures but it isn't given here, it doesn't mean you don't need to know anything about it!

Basic themes:

1. The primary topic of the course is information and its flow.

2. We try to understand this process at the MOLECULAR LEVEL. So the focus is on looking at the molecules and their interactions.

3. Specificity--most of the interactions have a high degree.

4. They are controlled by a number of weak interactions. Be aware of the advantages of this.

a. Often these interactions involve many proteins that assemble into a "protein machine" on DNA or RNA.

5. The combination of genetics and biochemistry is more than the sum of the parts.

Genetics—synthetic, looks at changes in the context of the entire cell
Biochemistry—analytic, divide-and-conquer--looks at the individual components and then at their interactions.

6. There is a strong interrelationship between mechanism and regulation: Once we understand the mechanisms, we can understand regulation. Conversely, studying regulation can help us get at the mechanisms. An example: studies of lac operon regulation led to the concept of mRNA.

7. Making and testing hypotheses—the intent in emphasizing this exercise is
a. to help understand where models come from
b. help you learn the models
c. help you learn the process of science

8. Critical Experiments--ones that test predictions—the goal of a critical experiment is to try to DISPROVE the hypothesis, not to prove it. Push the model hard, see how it holds up.

 

Specific points:

Genetics:

Know the meaning of several terms: dominance, recessiveness, epistasis.
How are epistasis tests used?
Compare and contrast classical genetics and reverse genetics.

Biochemical methodology:

Have a general idea of how various methods work and what information they give you.
Examples are footprinting, gel mobility assay, and the in vitro transcription system.
Understand clearly what I (as a biochemist) mean by an in vitro experiment.

Specific Protein-DNA interactions:

Specific interactions include a non-specific component. This usually provides most of the energy that favors binding.
Have some idea of the types of forces involved.
Energetics—wrong H-bonds are costly, more than the right ones being helpful.

Know about the common motifs found in DNA-binding proteins:
Helix-turn-helix
Zn finger (three types)
Leucine zipper
Helix-loop-helix

Most of these are platforms used to position a-helices in the major groove of DNA--that is, they establish a particular conformation of the protein that can interact with the DNA and can hold the a-helix stably in the major groove. You can review these with the RasMol scripts that were used in class.

The last two motifs are dimerization domains; they also allow the possibility of making heterodimers.

Most DNA-binding proteins bind to sites with dyad symmetry. Note that heterodimers often bind to sites in which the two halves differ (example is yeast a1:a2).

Details of molecular interactions: Most interactions occur in the major groove. They involve interactions between amino acid side chains and the sides of the base pairs. All four base pairs canbe distinguished in the major groove, but not in the minor groove. For the details, look at structures using RasMol.

Know the sizes of atoms and molecules: diameter of DNA helix, distance along the helix axis between base pairs, lengths of typical hydrogen bonds.

There is not a 1-to-1 correspondence between an amino acid and a base pair:

1. Many base pairs interact with >1 amino acid
2. Some amino acids interact with >1 base pair.
3. In different cases, a given base pair can interact with different amino acids, and conversely.

Mechanisms of transcription:

The transcription cycle is better understood in the case of prokaryotes than eukaryotes.

Prokaryotes:

1. RNA polymerase can be considered as a different enzyme at the various stages of the transcription cycle, because the reactions it catalyzes are very different.
2. Know the various steps in the cycle, and the types of reactions RNAP carries out.
3. Know what an open complex is. It forms in two steps, formation of the closed complex and transition to the open complex.
4. Know what constitutes a bacterial promoter; the fact that sigma gives the specificity; and how one might show experimentally that a protein is a sigma factor.
a. Know that other sigma factors exist; in particular, that 54 works somewhat differently, in that it readily allows closed complexes to form but an activator and ATP are needed to lead to open complex formation.
5. Transition to elongation mode
6. Elongation—probably the elongation complex is the same at each step (not the inchworm)
Sliding clamp model—at each step, interactions between the RNAP and the DNA and RNA are made and stabilize the elongation complex
7. Pausing: Elongation is not a steady process. Instead, RNAP often pauses for a period of time, then continues elongating. Pausing often occurs at stem-loop structures.
8. Termination: Pausing is an intermediate in the termination process.

Two types of terminators:
a. Rho-independent (or simple) terminators: Know their components
b. Rho-dependent terminators: Require rho protein. Rho binds to unstructured regions of RNA, and travels towards the RNA polymerase. Rho may act as an RNA helicase.

9. Interaction with activators: Know that different activators touch different parts of the transcription machinery. For example:

CAP touches two different parts of the a subunit

CI touches the s subunit

These can work in a synergistic fashion (a theme more common in eukaryotic regulation)

Eukaryotes:

1. Understand the relevant differences between eukaryotes and prokaryotes:
a. Eukaryotes have a nucleus
i. Transcription and translation are not coupled
ii. Proteins destined for the nucleus are targeted to that compartment.
b. Eukaryotes have chromatin
c. Eukaryotes have cell-type specific gene expression

2. Eukaryotes have three RNA polymerases; know the types of genes they transcribe.
3. Polymerases don't themselves recognize promoters. Instead, a protein machine made up of general or basal transcription factors assembles at the start point of transcription, and the polymerases enters the complex at a relatively late stage.

Polymerase II — makes mRNA; its action is extensively regulated.

Basal or general transcription machinery--assembles the pre-initiation complex (PIC) on a minimal promoter; generally this has been done in the absence of transcription factors.

There are two models for assembly: An ordered pathway, and holoenzyme. Know what each term means. How could workers have found evidence for an ordered pathway, if the holoenzyme model turns out to be correct?

TFIID—it has several subunits, the TATA binding protein (TBP) and several TAF's. TBP or TFIID binds first; then either the order pathway follows or the holoenzyme binds to bound TBP or TFIID.

TBP: Know how it was cloned. Be able to give a general overview of its structure in complex with the TATA box (look at the structure with RasMol).

 

Regulation:

Some general principles:

1. To qualify as regulation, the system must behave differently under different circumstances. There are several ways to make this happen:
a. Presence or absence of a regulatory protein
b. Presence or absence of a ligand
c. Alteration of the function of a regulatory protein by
i. Ligand binding
ii. Change in stability
iii. Post-translational modification
d. Order of events--dynamics rather than thermodynamics

2. A given protein can have different functions, depending on site of action and on circumstances; for instance, l CI can be an be an activator or repressor.

3. A given gene can have different regulatory controls, and these can have differing regulatory meanings. Examples:
a. pRM and pRE for transcription of lcI.
b. Multiple controls on the yeast HO gene.

4. Behavior of regulatory circuits: Autoregulation, positive and negative feedback, establishment vs. maintenance of a regulatory pattern.

5. Be able to distinguish between positive and negative regulation by analyzing the properties of non- inducible and constitutive mutants.

Prokaryotes:

Lambda:

Understand the l life cycle--two mutually-exclusive regulatory pathways can occur
Understand how the decision between the lytic and lysogenic pathways is determined--what are the roles of cII, Hfl, and cIII?
Understand how the regulatory circuitry stabilizes the regulatory decision once it is made.
Understand the functions of N and Q as anti-terminators. Understand that these proteins modify RNAP at particular sites (nut or qut), not at the sites of action (terminators).
Understand the process of prophage induction.

Trp attenuation:

This mechanism is unusual in being dynamic--what controls events is what happens first--a given RNA secondary structure only has to persist long enough for RNAP to terminate or pass the attenuator.
Role of alternate stem-loops in decision.
Role of pausing at the 1:2 stem-loop—it lets ribosome catch up.
Understand the distinction between transcription and translation in this system, and the role that translation of the leader peptide plays.

Eukaryotes:

Terminology:

trans-acting factors (activators and repressors) — proteins that bind to sites on any chromosome
cis-acting sites — sites to which trans-acting factors bind; they affect only the chromosome to which they are physically attached
haploid—having one copy of the genome
diploid—having two copies of the genome

Cis-acting sites:

Two main types: Promoters and associated elements, and enhancers
Promoters consist of a TATA box; associated elements generally lie close to the TATA box and have a fixed relationship with it.
Enhancers can lie at variable distances, and in either orientation; they are thought to function by looping, which brings factors bound to them close to the TATA box.

Trans-acting factors:

Have some idea how they are identified, purified, cloned, and characterized.

Properties of eukaryotic activators:

1. Often have a modular or domain organization
2. Can involve different kinds of DNA-binding motifs
3. Activation domains can be of different types
4. Some factors do not bind DNA, but instead bind to DNA-bound proteins. Such proteins are called coactivators.

Function of activators:

Activators work by two main mechanisms:
1. They antagonize the repressive effects of chromatin (see below)
2. They recruit the basal transcription machinery into the PIC

Recruitment:

1. Activators generally act by recruiting components of the basal machinery into the PIC.
2. This interaction can be with different components of the basal machinery—mediator or TAF's—it may be different in yeast (mediator) and higher eukaryotes (TAF's) (not yet understood).
3. A given activator may interact with more than one component.
4. Have an idea of how the modular organization of typical activators was established.

Synergism:

Several different activators, or several molecules of the same activator, can give much greater than additive effects on stimulating transcription. Perhaps they are making multiple contacts.

Repression in eukaryotes:

There are several ways it can work: Inhibition of DNA binding by another factor; interference with activation; interference with assembling a pre-initiation complex; and histone deacetylation.

Co-activators and co-repressors:

Some regulatory proteins do not bind to DNA directly, but to other components of the transcription machinery. These are called co-activators if they activate, and co-repressors if they repress. Examples of co-activators are the TAF's and CBP.

Yeast mating types and mating-type switching:

Two regulatory problems:
How is the mating type switched?
How does the MAT locus control the mating type?

Switching: understand the cassette model and the role of HO in switching.

Understand how MAT controls mating type:

Three different types of genes: a, a , haploid-specific

Understand the specific roles of a1, a2, and a1 in controlling these.

Action of a2:MCM1: It recruits SSN6-TUP1, which are co-repressors; in turn, they somehow act to turn off a-specific genes, which are located near binding sites for a2:MCM1.

 

Chromatin:

Chromatin condenses the DNA and makes it unavailable for transcription.

Have an idea of the structure of the nucleosome (link to PDB file): The DNA is wrapped tightly around the outside of a histone core, almost two turns, tightly bent, histone tails protruding outwards from the surface.

One major role of activators is to remove the general repression conferred by nucleosomes. Conversely, some negative regulation favors it.

Cells have active control over the stability of chromatin. Chromatin remodeling machines "remodel" the chromatin to make it more (or less) accessible to the transcription machinery. An example is the SWI/SNF complex. The Holstege et al (1998) paper described how some genes are turned on, and some turned off, in a swi2- mutant. It isn't clear at the molecular level what changes are caused by chromatin remodeling; perhaps it simply repositions the nucleosomes, or in some poorly understood way "loosens" their structure.

In some cases, nucleosomes are precisely positioned, in part because the DNA sequence favors certain locations and in part as a result of specific regulatory events.

Histone acetylases (HAT's) and deacetylases (HDAC's):

Histones can be acetylated on lysine residues.
1. Acetylation generally leads to greater gene expression; deacetylation to reduced gene expression.
2. Two ideas for how this might work:
a. Reduce positive charge of histones, giving weaker DNA binding
b. Destabilize interactions between nucleosomes

3. HAT's and HDAC's are targeted to specific sites; understand how. Understand the advantage of this in regulating gene expression.ere are several methods in common use for analyzing specific protein-DNA interactions. It is important to understand these methods, and to be aware of the capabilities and limitations of each method.

 

Regulation in higher eukaryotes:

NOTE: Here are some general principles, for your own information. I won't ask specifically about these on the exam.

For several reasons, it is more complicated in metazoa than in yeast:
a. Positive regulation is thought to overcome rate-limiting steps. This step might not be the same for all promoters (this may apply in yeast as well).
b. Many regulated promoters contain binding sites for constitutively expressed factors. At many regulatory sites, several proteins assemble into a large protein machine ("enhanceosome").
c. There are multigene families of closely related DNA binding proteins with similar or identical sequence specificity. E.g., Jun and Fos are members of two families; AP-1 is a Jun-Fos heterodimer, but probably other forms of AP-1 can contain different family members; likely, many or all of these bind to the same site (TGACTCA). Consequence: It doesn't suffice to identify binding sites; that doesn't tell you what is bound there.
d. Different proteins can bind to the same or closely related sequence. Maybe other factors help confer specificity.


BIOC/MCB 568 -- University of Arizona
http://www.biochem.arizona.edu/classes/bioc568/bioc568.htm
Last modified August 18, 2010
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