Chapter 19- Gene Regulation in Eukaryotes

Eukaryotic Gene Regulation

-almost all cells in an organism are genetically identical, but cells will only make proteins for their function to avoid wasting energy

-differences between cell types result from differential gene expression

-gene expression is regulated at many stages (some genes are always "on")

Typical Histone Structure

-histones have tails with (+) charge which when combined with negatively charged DNA, making DNA tightly wrapped and less accessible for transcription

Histone/Chromatin Modification- Acetylation

-acetyl groups are added to tails (which blocks the + lysine charge meaning it stays open and promotes transcription)

-loosens chromatin and promotes transcription

Histone/Chromatin Modification- Methylation

-tightens chromatin and inhibits transcription

OR

-loosens chromatin and promotes transcription

DNA Methylation as Gene Expression Regulator

-adding methyl groups to certain bases in DNA (usually cytosine) typically reduces transcription, especially if added to promoter regions

-CpG regions targeted for methylation purposes (cytosine directly followed by a guanine)

Cis-Acting Regulatory Elements

-located on the same DNA strand as whichever one they are controlling

-not transcribed themselves (are not located within transcription regions)

Cis-Acting Regulatory Elements- Promoter

-located very close to the gene it controls, usually contains the TATA box

-proximal control elements are located close to the promoter

-recognition site for RNA polymerase

-only allows basal levels of transcription

Cis-Acting Regulatory Elements- Enhancer

-groups of distal control elements (can be upstream/downstream and very far away)

-distal control elements are located farther away from the promoter or within introns

-a single gene can have one enhancer or multiple enhancers

-a single enhancer can be recognized by multiple factors

Identifying Enhancers and Promoters Using GFP

-GFP from jellyfish acts as a reporter

-more green fluorescence, more gene expression

-less green fluorescence, less gene expression

Creating a Reporter Construct

-create a recombinant DNA molecule: potential reporter region + GFP gene

-create a transgenic organism that has the recombinant DNA reporter construct

-if the potential reporter region is a promoter, GFP expression will be at basal levels

-if the potential reporter region is an enhancer, GFP expression will be at higher levels

Determining Promoters and Enhancers

-no promoter, no transcription (-)

-no enhancer, less fluorescence than the original (+ compared to +++)

Trans-Acting Regulatory Elements

-transcription factors bind to control elements (proximal and distal)

-can inhibit or activate gene expression

-general transcription factors (all/most genes) vs specific transcription factors (single/few genes with similar function)

-TFs bind to enhancers and DNA bending allows them to contact promoter proteins

Trans-Acting Regulatory Elements- Basal Transcription Factors

-bind to promoter regions

-assists binding of RNA polymerase to the promoter region (like an airplane marshaller)

-TATA box-binding proteins (TBPs) recruit TBP-associated factors (TAFs) to the promoter

Trans-Acting Regulatory Elements- Activators

-bind to enhancer regions and enhance transcription above basal levels

-DNA bends if activators are bound to far away enhancer regions

-two main functions:

1. recruit basal factors and RNA polymerase II to the promoter

2. recruit co-activators that open local chromatin structure to allow transcription

Trans-Acting Regulatory Elements- Repressors

-bind to specific DNA sites near the gene and prevent gene expression

-recruits co-repressors to enhancer regions to...

1. interact with basal complexes and block binding to the promoter

2. modify histone tails to close chromatin structure

Identifying Transcription Factors Using GFP

-create a transgenic organism that has this GFP reporter and introduce the loss of function mutation in candidate genes

-if the loss-of-function mutation is within an activator, the reporter will only show basal expression (lower GFP)

-if the loss-of-function mutation is within a repressor, the reporter may show higher levels of GFP expression

Transcription Factor Conclusions Using GFP

-take away the repressor -----> higher GFP

-take away the activator ------> lower GFP

Trans-Acting Regulatory Elements: Flexibility

-some transcription factors can act as either activators or repressors (depends on the enhancer region/gene and other transcription factors that also bind)

-ex: drosophila melanogaster with dorsal typically acting as an activator

-if dorsal interacts with certain TFs, groucho is recruited leading to transcriptional repression

Indirect Repression

-indirect repressors can block transcription factors by interfering with activators through the following mechanisms:

-competition of DNA binding site

-quenching of activator function

-cytoplasmic sequestration

-drain activator concentration

Combinatorial Control of Gene Activation

-each enhancer ~10 control elementals, with each control element ~1-2 specific transcription factors

-gene regulation is controlled by a combination of control elements in an enhancer and the activator proteins present

-groups of genes can be controlled simultaneously by all having the same combination of control elements (metabolic pathways, etc.)

Insulators

-organize chromatin so that enhancers have access to only particular promoters

-located in between a promoter and enhancer ---> blocks interaction

-enhancers cannot interact with promoters on a different loop

Post-Transcriptional Regulation- RNA Processing

-alternative splicing creates different mRNA molecules from the same pre-mRNA transcript

-a variety of proteins can come from one gene

Post-Transcriptional Regulation- mRNA Degradation

-mRNA transcripts are temporary and their stability partially relies on alteration of the ends.

-UTR of the 3' end may contain a signal to control lifespan of mRNA transcript

Post-Transcriptional Regulation- Initiation of Translation

-regulatory proteins can bind to the 5' UTR or '3 UTR and control ribosome activity

-some activate translation, while others block translation

Post-Transcriptional Regulation- Protein Processing

-newly made proteins often need modifications to become "active"

-addition of carbohydrate or lipid groups, phosphorylation, etc.

Post-Transcriptional Regulation- Protein Degradation

-protein lifespan is tightly controlled

-ubiquitination flags proteins for the proteasome

Non-coding RNAs

-only a small fraction (2%) of the genome may be transcribed into noncoding RNAs (ncRNAs)

-a significant amount of the genome may be translated into noncoding RNAs

MicroRNAs (miRNAs)

-small single-stranded RNA molecules that can bind to mRNA

-degrade mRNA or block its translation

Small Interfering RNAs (siRNAs)

-small single-stranded RNA molecules that can bind to mRNA

-block gene expression similar to miRNAs (RNAi)

Long Non-coding RNAs (lncRNAs)

-large single-stranded RNA molecules that can bind to DNA

-involved in condensing chromosomes for inactivation (X)