Transcriptional Regulation Notes

Architectural Regulators

• Architectural regulators bind to DNA at architectural regulator-binding sites.
• They alter the structure of DNA/chromatin.
• This indirectly facilitates transcription by making it easier for distant transcriptional activators to reach the promoter.

Cofactors

• Cofactors (coactivators and corepressors) are proteins that do not bind DNA directly.
• They facilitate interactions that activate or repress transcription.

Insulators

• Insulators are cis-elements that block activators and repressors from affecting transcription from one side of the insulator to the other.
• Also called boundary elements.
• The mechanism is still unclear but probably involves blocking DNA looping.

Effectors

• Effectors are small molecules that can bind to transcriptional regulators and affect their function.
• Effectors can repress transcription (inhibitors) or activate transcription (inducers).
• Effectors are sometimes metabolites produced by the gene they regulate and can act in a positive or negative feedback loop.
• Examples of effectors interacting with activators include:
  • Inhibiting transcription
  • Activating transcription

Fructose-2,6-bisphosphate (F-2,6-BP)

• Strong allosteric activator of phosphofructokinase (PFK).
• High levels of Fructose-6-phosphate (F-6-P) lead to the production of F-2,6-BP, which activates PFK to increase the rate of glycolysis.
• This is a feed-forward activation mechanism.

The lac Operon

• A classic example of gene expression regulation in bacteria.

lac Operon Components

• $lacZ$, $lacY$, and $lacA$ encode proteins that metabolize lactose.
• $lacI$ and $lacO$ regulate when the lac operon is expressed.
• $lacI$ and $lacO$ are important for repressing lac operon expression.
• Mutations in $lacI$ or $lacO$ lead to uncontrollable expression of Lac genes.

lacI and lacO

• $lacI$ encodes the lac repressor protein.
• $lacO$ is the binding site for the lac repressor (called an Operator).
• $lacO$ acts in cis.
• The $lacI$ product acts in trans.
• The lac repressor binds to the operator sequence to block Lac expression.

lac Operon and Lactose

• The lac operon can sense when lactose is present.
• In the presence of lactose, the lac repressor is blocked, allowing transcription.
• In the absence of lactose, the lac repressor blocks transcription.
• Allolactose is the inducer.
• An inducer (a type of effector) binds to the lac repressor, blocking its function and allowing lac expression.

Allolactose

• Allolactose is a byproduct of lactose metabolism by the lac operon.
• A low basal level of the lac genes is always expressed, producing enough allolactose when lactose is present to block the lac repressor.
• When allolactose is produced, it blocks the lac repressor, allowing lac expression.

How the lac Repressor Works

• There are 3 lac repressor binding sites on the lac operon: $O1$, $O2$, and $O3$.
• $O1$ is the original and only required site.
• The lac repressor binds to $O1$ and either $O2$ or $O3$, creating a loop.
• The loop physically blocks RNA polymerase.

Glucose and the lac Operon

• Lactose is not as efficient an energy source as glucose.
• When glucose is abundant, there’s no need to express the lac genes, even if lactose is present.
• Cyclic AMP (cAMP) is over-produced when glucose is absent, but turned off when glucose is abundant.
• When glucose is not there, cAMP is how the cell tells the Lac operon and other sugar metabolizing operons that it’s time to turn on.
• cAMP is an inducer of Lac expression.
• cAMP binds to a protein called cAMP responsive protein (CRP) to activate it.
• CRP-cAMP binds to the Lac promoter to activate transcription (i.e. it’s an activator).
• In the absence of lactose, the lac repressor is still bound, preventing CRP-cAMP from turning on the lac genes when there’s nothing for them to metabolize.

cAMP-CRP

• In the presence of glucose, there’s no cAMP-CRP, so no effect on the lac operon.
• In the absence of glucose, cAMP-CRP can bind to the Lac promoter, but if lactose is also absent, the lac repressor blocks cAMP-CRP.
• When glucose is high, there’s no free cAMP-CMP. If lactose is also present, the lac repressor is blocked, but the positive signal given by cAMP-CMP is not there to help and only very low transcription can occur.
• When glucose is low, cAMP-CMP bind to the lac promoter. If lactose is high, then the lac repressor is unbound and high transcription

Common Motifs in Transcription Factors

• Helix-turn-helix Motif
• Leucine Zippers
• Zinc Fingers

DNA Binding Proteins

• Regulatory binding site sequences are often inverted repeats (CANNTG).
• DNA binding proteins are often dimers.

Helix-turn-helix motif

• Recognition helix – One helix (red) fits into major groove
• Most DNA-binding motifs recognize the major groove because it has more features to interact with (hydrogen bond donors/acceptors, etc.) than the minor groove.

Leucine Zipper

• A motif consisting of several leucines spaced about 7 amino acids apart
• Zipper regions hold two subunits together, not involved in DNA binding, but are linked to a DNA binding region.

Zinc Fingers

• Zinc finger motifs are held together by Zinc ions.
• Each motif has a DNA recognition helix.
• Each motif is weak, but can be stacked together (beyond dimerizing), with each motif acting like a finger, gripping the DNA by the major groove.
• Zinc ion stabilizes structure but is not directly involved in DNA recognition.

Chromatin Structure

• Chromatin structure is the first barrier to transcription
• Heterochromatin – region of chromatin associated with gene silencing.
• Euchromatin – a more accessible form of chromatin to the transcription machinery. Sensitive to DNaseI digestion.
• Chromatin remodeling – changing chromatin structure to make it more or less accessible
• “Closed” versus “Open” chromatin is a better way to think about it.

Regulating Chromatin Structure

• Chromatin remodeling is controlled by at least three mechanisms:
  • Nucleosome repositioning on the DNA – Chromatin remodeling complexes shift the position of nucleosomes so that the promoter is no longer is wrapped around a nucleosome.
  • Histone variants – Histone variants can be inserted to substitute for the main histone subunits, altering DNA binding ability.
  • Histone modification – Histones are modified by acetylation or methylation, altering DNA accessibility.

Histone Modifications

• Histone proteins make up the core of the nucleosome. Their tails can be modified to regulate accessibility of the DNA
• Acetylation – almost always associated with increasing accessibility and activating transcription
  • Specific lysines on histone tails are acetylated (e.g. H3K9ac)
  • HATs (histone acetyltransferases) and HDACs (histone deacetylases) regulate histone acetylation
• Methylation – mostly associated with silencing transcription, but in some cases activation. Usually tri-methylated.
  • Example: H3K9me3

Nucleosome Repositioning

• DNA is wrapped tightly around nucleosomes
• In order to allow access to those sequences to transcription machinery, the nucleosomes must be shifted
• SWI/SNF – a chromatin remodeling complex that can reposition nucleosomes to free up the promoter
• SWI/SNF is recruited to specific regions by transcription activators, thus the transcription activators must bind to their DNA sequences first

Turning on a Gene

1. Transcription factors specific to a gene bind to regulatory sites near or far from the promoter of that gene
2. HATs are recruited to acetylate histone tails
3. Chromatin remodeling complexes (SWI/SNF) are also recruited and reposition the nucleosomes to expose the promoter
4. The chromatin structure around the gene (particularly the promoter) goes from a closed configuration to an open configuration
5. Transcription factors recruit mediator, and architectural regulators bend the DNA into loops, collectively bringing these complexes in close proximity to the promoters
6. All of this begins to recruit the transcription machinery (aka general transcription factors and RNA pol) to assemble on the promoter
7. Transcription initiation occurs
8. Transcription terminates
9. HDACs are recruited to de-acetlyate histone tails.

Turning off a Gene

• Unlike bacteria (think lac repressor), eukaryotic repressors generally only turn off transcription after it’s already started.
• Repressors/co-repressors kick off activators and inhibit RNA pol, turning off transcription
• Histone deacetylation by HDACs leads to a closed chromatin configuration, sealing the closed state

DNA Methylation

• Direct DNA methylation inhibits gene promoters
• Cytosines of CG pairs (also called CpG) can be methylated
• DNA Methylation at the promoter is almost always associated with the repression of gene expression and closed chromatin

1. Methylated DNA could inhibit transcriptional machinery from binding to the promoter
2. Methylated DNA could recruit HDACs and other complexes associated with closed chromatin configurations

RNA interference (RNAi)

• siRNA – short interfering RNA. About 21-27 nucleotides long. Sequence is the reverse complement of transcript and will hybridize with it to form double-stranded RNA.
• Anti-sense RNA – an RNA whose sequence is the reverse complement of another RNA (sense RNA). Anti-sense and sense RNA will hybridize to form double-stranded RNA
• microRNA (miRNA) – RNAs produced naturally by cells to regulate gene expression. After processing, they’re around 20-22 nucleotides
• The gene whose mRNA is bound to by siRNA becomes silenced

miRNA Discovery

1. miRNAs are transcribed (pri- miRNAs) and form hairpins
2. Drosha chops them into short segments of hairpins (pre-miRNA)
3. Pre-miRNAs are exported and loaded onto the RISC complex
4. Dicer cleaves the hairpin to create double-stranded RNA (miRNA duplex)
5. An RNA helicase separates the two strands, and the sense strand is removed
6. The final mature mRNA is single-stranded anti-sense, and still within the RISC complex

How miRNAs Silence Genes

1. Perfect or near perfect complementarity
   Forms dsRNA structure and the mRNA is rapidly degraded
2. Partial complementarity
   RISC complex remains bound to miRNA and mRNA, and translation is physically blocked. Eventually the mRNA is degraded

miRNA Targets

• Usually 8mers in the 3’ UTRs of mRNAs (but not always)
• 1 miRNA can recognize many, many target genes
• There are estimated to be around 800 miRNA genes (e.g. miR-142, miR-181a)
• miRNAs likely have more subtle regulatory effects on an individual gene but can impact a lot of genes at once
• Each gene can have binding sites for multiple miRNAs
• ~5000 human genes are targeted by at least 1 miRNA

miRNA vs. siRNA

• siRNA - Exogenously added (viruses, researchers)
• shRNA – short-hairpin RNA. Created by researchers. When placed into a DNA plasmid, once transcribed will form a short-hairpin, which will be processed by the miRNA machinery.
• siRNAs are typically created as single-stranded or double-stranded RNA, whereas shRNA starts out as a DNA sequence.
• miRNA - Encoded by genome

Learning Goals

• Understand the difference between cis and trans and how to identify which is which experimentally
• Know the names of each of the major classes of trans-acting factors and cis-acting elements
• Understand how the lac operon is regulated and be able to analyze experimental data on mutations of components of the lac operon
• Know the basics of the 3 structural motifs
• Understand how changes in chromatin make genes more or less likely to be transcribed
• Be familiar in the basic steps and order of steps in turning on a gene, and off a gene
• Be familiar with the experiments of Mello and Fire
• Understand how microRNAs are produced and what each factor does in the process