BS 161: Chapter 19 Part I (Eukaryotes) Review

Histone acetylation

(addition of an acetyl group, —COCH3, the enzymes involved are called Histone acetyl transferases (HATs)) and deacetylation appear to play a direct role in the regulation of gene transcription.

Chromatin modifications: key concept

*Chromatin modifications affect the availability of genes for transcription.*

Differential gene expression

*Differential gene expression is the expression of different genes by cells with the same genome* A typical human cell probably expresses about 20% of its genes at any given time. Highly specialized cells, such as nerves or muscles, express only a tiny fraction of their genes. Although all the cells in an organism contain an identical genome, the subset of genes expressed in the cells of each type is unique.

Eukaryotic cells package their DNA with __________.

*Eukaryotic cells package their linear DNA with proteins as one molecule per chromosome.*

Eukaryotic gene expression: key concept

*Eukaryotic gene expression can be regulated at any stage.*

Gal4

*Gal4 works a lot like the CRP (cAMP response protein) in bacteria, in that it can help recruit in the general transcription factors for efficient transcription initiation. How do the Distal enhancers work? They can be thousands of nts away from the promoter!!*

Inactive DNA

*Inactive DNA is generally highly methylated compared to DNA that is actively transcribed.* For example, the inactivated mammalian X chromosome in females is heavily methylated

Other chemical groups and histone tails

*Several other chemical groups, such as methyl and phosphate groups, can be reversibly attached to amino acids in histone tails.*

Chromatin modification inheritance

*The chromatin modifications just discussed do not alter the DNA sequence, and yet they may be passed along to future generations of cells.*

Harold Weintraub and Mark Groudine: Experiment

*They took chicken blood cells - looked specifically at 2 genes.* 1) β-globin - hemoglobin - red blood cells - hemoglobin - highly expressed. 2) Ovalbumin - protein makes up the egg whites. Not expressed in red blood cells. The red blood cells were gently lysed and treated with DNase. Analyzed the DNA for these two genes.

Activator: 2 structural elements

A *DNA binding domain* and one or more *activation domain* Activation domains bind other regulatory proteins or components of the transcription machinery to stimulate transcription.

Enhancers (distal control elements)

A given gene may have multiple enhancers (each enhancer with a specific set of control elements), each active at a different time or in a different cell type or location in the organism. Interactions between enhancers and specific transcription factors called activators or repressors are important in controlling gene expression.

Acetylated histones

Acetylated histones grip neighboring nucleosomes less tightly, providing easier access for transcription proteins in this region.

Acetylation of the lysine in the histone tales

Acetylation of the lysines in the histone tails reduces their positive charge, so they interact with the DNA less tightly. Histone acetyl transferases (HATS) are activators of transcription. Likewise, enzymes that remove the acetyl groups (Histone deacetylases (HDACS)) would be considered Repressors of transcription.

Example #1: Proximal control elements - Yeast Galactose Metabolism Figure

All 5 enzymes for galactose metabolism have the same promoter proximal element. That means they can all bind the same transcription factor. Only these 5 enzymes have this promoter proximal element that can bind Gal4. So when Gal4 is activated, all 5 enzymes are turned on.

Activator

An activator is a protein that binds to an enhancer to stimulate transcription of a gene.

If the bound Regulatory proteins are thousands of bases away, How do they do anything at the promoter?

Bending of the DNA enables enhancers to influence a promoter hundreds or even thousands of nucleotides away.

Histone modifications regulate gene transcription

Chemical modifications of the histones and DNA of chromatin play a key role in chromatin structure and gene expression.

Regulation of transcription: Control elements

Control elements are noncoding DNA segments that regulate transcription by binding certain proteins. These control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types.

There are four main levels of packaging: level 1

DNA is wrapped twice around a group of histone proteins (the core histones). The core histones are made up of eight histone proteins, two each of histones H2A, H2B, H3 and H4. Level one is often called the "beads on a string" and is ~10 nm in width. In general, the genome packaged in this conformation (level 1) is transcriptionally active.

DNA methylation: Key concept

DNA methylation reduces gene expression

Distal control elements

Distal control elements (enhancers), may be thousands of nucleotides away from the promoter or even downstream of the gene or within an intron.

Distal regulatory sequences are called

Enhancers

Example #1: Proximal control elements - Yeast Galactose Metabolism terms: monocistronic

Enzymes - are monocistronic. Unlike Bacteria, most eukaryotics do not have polycistronic genes. They are monocistronic. Each gene is regulated by its own promoter. They can be separated by a lot of space, they can be on different chromosomes. Q: How do you coordinate expression of this group of genes? A: Find same promoter proximal elements upstream of all 5 genes. 1 Transcription Factor - called Gal4 - Activator

Epigenetic variations

Epigenetic variations may explain why one identical twin acquires a genetically based disease, such as schizophrenia, while another does not, despite their identical genomes. In addition, we now know that certain environmental factors (exposure to BPA for example) may influence DNA methylation and play significant roles in epigenetic variation.

Repressors

Eukaryotic genes also have repressors to inhibit the expression of a specific gene. Eukaryotic repressors can inhibit gene expression by blocking the binding of activators to their control elements or to components of the transcription machinery. Other repressors bind directly to control-element DNA, turning off transcription even in the presence of activators.

Prokaryotic mRNA molecules are typically degraded after only a few minutes. But eukaryotic mRNA.....

Eukaryotic mRNAs typically last for hours, days, or weeks. For example, in red blood cells, mRNAs for hemoglobin polypeptides are unusually stable and are translated repeatedly. Other mRNAs may last for only minutes.

Example #1: Proximal control elements - Yeast Galactose Metabolism

Galactose is a sugar - yeast can use it, requires several enzymes (5) to metabolize. No galactose - yeast don't synthesize these enzymes. Add galactose - increase synthesis of all 5 enzymes ~1000 fold.

Post-transcriptional mechanisms

Gene expression may be blocked or stimulated by any post-transcriptional step. By using regulatory mechanisms that operate after transcription, a cell can rapidly fine-tune gene expression in response to environmental changes, without altering its transcriptional patterns.

General transcription factors

General transcription factors are essential for the transcription of all protein-coding genes and help in the recruitment of RNA polymerase. However, the interaction of general transcription factors and RNA polymerase II with a promoter *usually leads to only a slow rate of initiation* and the production of few RNA transcripts. Bind to other proteins or to a sequence element within the promoter called the TATA box.

Genes of densely condensed heterochromatin

Genes of densely condensed heterochromatin are usually not expressed, presumably because transcription proteins cannot reach the DNA.

Alternative RNA splicing

In *alternative RNA splicing*, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. Regulatory proteins specific to a cell type control intron-exon choices by binding to regulatory sequences within the primary transcript.

Specific transcription factors

In eukaryotes, high levels of transcription of particular genes depend on the interaction of control elements with *specific transcription factors*.

Gene expression and transcription

In most organisms, the expression of specific genes is most commonly regulated at transcription, often in response to signals coming from outside the cell. For this reason, the term gene expression is often equated with transcription. However, with their greater complexity, eukaryotes have opportunities for controlling gene expression at additional stages.

Polycistronic organization: operons

In prokaryotes, coordinately controlled genes are often clustered into an operon with a single promoter and other control elements upstream. The genes of the operon are transcribed into a single mRNA and translated together (Polycistronic organization!). Very few eukaryotic genes are organized this way

DNA methylation

In some species, DNA methylation is responsible for the long-term inactivation of genes during cellular differentiation. Once methylated, genes usually stay that way through successive cell divisions in a given individual. Methylation enzymes recognize sites on one strand that are already methylated and correctly methylate the daughter strand after each round of DNA replication.

Epigenetic inheritance

Inheritance of traits by mechanisms not directly involving the nucleotide sequence is called *epigenetic inheritance*.

Regulation of Gene expression in Eukaryotes

Like unicellular organisms, the tens of thousands of genes in the cells of multicellular eukaryotes are continuously turned on and off in response to signals from their internal and external environments. Gene expression must be controlled on a long-term basis during cellular differentiation.

*Organization of eukaryotic genes*: Coordinate gene expression

Most eukaryotic genes coding for the enzymes of a metabolic pathway are scattered over different chromosomes. Coordinate gene expression in eukaryotes depends on the association of a specific control element (or combination of control elements) with every gene of a dispersed group. A common group of transcription factors binds to all the genes in the group, promoting simultaneous gene transcription.

Many proteins must undergo chemical modifications before they are functional

Phosphate groups activate regulatory proteins. Sugars are added to cell-surface proteins, which are then transported to their target destination in the cell.

Post-transcriptional mechanisms: Key Concept

Post-transcriptional mechanisms also play supporting roles in the control of gene expression.

RNA processing in gene regulation

RNA processing in the nucleus and the export of mRNA to the cytoplasm provide opportunities for gene regulation that are not available in prokaryotes.

Chromatin-modifying proteins

Recruitment of chromatin-modifying proteins seems to be the most common mechanism of repression in eukaryotes.

Experiments by Harold Weintraub and Mark Groudine

Simple experiments by Harold Weintraub and Mark Groudine first suggested that chromatin packing could affect gene expression. They Used the enzyme, DNAse I. DNAse I very efficiently degrades the DNA if it is stripped of proteins. It is much less efficient at cutting highly packaged chromatin.

Activators and repressors in chromatin structure

Some activators recruit proteins that acetylate histones (HATs) near the promoters of specific genes, promoting transcription. Some repressors recruit proteins that deacetylate histones (HDACs), reducing transcription or silencing the gene.

Proximal control elements

Some control elements, named proximal control elements, are located close to the promoter.

Four main levels of packaging: level 2

The "tails" of the core histones and Histone 1 can stack the nucleosomes together in a more tightly coiled conformation (30 nm in width).

Four main levels of packaging: level 3

The 30 nm fiber can then be further coiled into a 300 nm diameter fiber

Experiments by Harold Weintraub and Mark Groudine: Results

The DNAse had degraded the β-globin gene (found in fragments) while the Ovalbumin gene was intact (not degraded). They did this analysis on a number of genes - clear, DNAse I had access to the DNA for those genes that were being actively transcribed. Whereas, inactive genes were much more protected from the enzyme.

Histone proteins in eukaryotes

The Histone proteins are highly conserved in Eukaryotes and are rich in the amino acids lysine and arginine. These amino acids have R-groups that are positively charged and can interact with the negative charges of the phosphates along the backbone of the DNA.

N-terminus

The N-terminus of each histone molecule in a nucleosome protrudes outward from the nucleosome (Histone tails). These histone tails are accessible to various modifying enzymes, which catalyze the addition or removal of specific chemical groups.

Control of transcription: key concept

The control of transcription in eukaryotes depends on the binding of activators to DNA control elements.

The differences between cell types are due to ____________.

The differences between cell types are due to *differential gene expression*, the expression of different genes by cells with the same genome.

Post-translational regulation

The final opportunities for controlling gene expression occur after translation. Often, eukaryotic polypeptides are processed to yield functional proteins. For example, cleavage of pro-insulin forms the active hormone.

Translation presents an opportunity for the regulation of gene expression

The initiation of translation of an mRNA can be blocked by regulatory proteins that bind to specific *sequences or structures* within the 5' UTR (untranslated region) of the mRNA, preventing ribosome attachment.

Life span of an mRNA molecule

The life span of an mRNA molecule is an important factor in determining the pattern of protein synthesis.

Chromatin modifications

The location of a gene's promoter relative to nucleosomes and to the sites where the DNA attaches to the chromosome scaffold can affect whether the gene is transcribed.

Histone code hypothesis

The recent discovery that modifications to histone tails can affect chromatin structure and gene expression has led to the *histone code hypothesis*. This hypothesis proposes that specific combinations of modifications, rather than the overall level of histone acetylation, determine chromatin configuration. Chromatin configuration in turn influences transcription.

How do these enhancers function to aid transcription?

They bind to regulatory proteins

Genomic imprinting

This methylation pattern accounts for *genomic imprinting*, in which methylation turns off either the maternal or paternal alleles of certain genes at the start of development.

Transcription factors

To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called *transcription factors.*

Fine-tuning transcription initiation

Transcription initiation is controlled by proteins that interact with DNA and with each other.

Regulation of transcription

While Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more available or less available for transcription, eukaryotes also have multiple DNA elements (specific sequences of DNA that can bind specific proteins) that work together to *regulate transcription*.

Methyltransferases

While some enzymes methylate the tails of histone proteins, other enzymes (methyltransferases) methylate certain bases in DNA itself, usually cytosine.

Chromatin

the DNA/protein complex

Four main levels of packaging:

these can be further coiled (700nm in diameter) and finally the chromosomes can be fully condensed as observed during mitosis (1400 nm in diameter).