Lecture 9 Chapter 6Gene expression and regulation

IINeal Stewart

Focus questions

• How important are cis-regulatory elements and trans-acting factors in gene regulation?

• What are the control points that can regulate gene expression?

Transcription revisited

Promoter elements not required for transcription initiation

• CAAT box – usually located at -70 to -80 within the promoter

• GC box

• Other gene-specific elements (light-responsive, nutrient-responsive, etc.)

• Enhancer elements

What are some biological roles of transcription factors?

• Basal transcription regulation – general transcription factors

• Development

• Response to intercellular signals

• Response to environment

• Cell cycle control

The CRT/DRE response element responds to dehydration and cold-induced

transcription factors (CBF)

Figure 6.7

Transcription factors

Figure 6.8

Enhancer can work from downstream and upstream region

Enhancers

• Their location is not fixed. Location could be in the upstream or downstream DNA, in intron, exon or in the untranslated region.

• They enhance transcription by acting on promoter in cis (typically)

• Each enhancer has its own binding protein. These proteins are trans-regulatory activating factors

• Sequence of enhancers is variable.

• Enhancers regulate tissue-specific and temporal expression of genes.

TATA binding protein (TBP) transcription factor

Wikipedia.com

DNA-binding domains allow transcription factors to bind directly to a cis-regulatory element

Helix-loop-helix

Zinc finger domain

Leucine zipper domain

Extreme trans-acting effectors of transcription: TAL effectors

• From plant pathogenic bacteria Xanthomonas

• Secreted by bacteria when they infect

• Transcriptional activator-like (TAL) effectors bind with plant promoters to express genes beneficial for the bacteria

http://www.sciencemag.org/content/333/6051/1843/F2.large.jpg

Repression of transcription

TFs that act as repressors

Some trans-acting elements prevent transcription

Introducing RNAi

http://www.youtube.com/watch?v=H5udFjWDM3E&feature=related

What is a microRNA (miRNA)?What is a microRNA (miRNA)?Controlling gene expression post-transcriptionally.Controlling gene expression post-transcriptionally.

microRNA is an abundant class of newly identified small microRNA is an abundant class of newly identified small non-coding regulatory RNAs.non-coding regulatory RNAs.

Major characteristics of miRNAs:

• 18-26 nt in length with a majority of 21-23 nt

• non-coding RNA

• derived from a precursor with a long nt sequence

• this precursor can form a stem-loop 2nd hairpin structure

• the hairpin structure has low minimal free folding energy (MFE) and high MFE index

Slide courtesy of Baohong Zhang, East Carolina Univ

miRNA regulates plant development

WT miRNA

miRNA 156

increasing leaf initation, decreasing apical dominance, and forming bushier plant.

miRNA 164

stamens are fused together.

miRNA 172

sepal and petal disappeared.

miRNA 319

Leaf morphology

Slide courtesy of Baohong Zhang, East Carolina Univ

Small interfering RNAs inhibit expression of a homologous gene

Biogenesis of miRNAsBiogenesis of miRNAs

Bartel, 2004. Cell.

Plant Animal

Post-transcriptional gene regulation

Two major molecular mechanisms

Mechanisms of miRNA-mediated gene regulationMechanisms of miRNA-mediated gene regulation

Zhang et al. 2006. Developmental BiologySlide courtesy of Baohong Zhang, East Carolina Univ

Mary-Dell Chilton

• Undergrad and PhD University of Illinois• Postdoc with Gene Nester and Milt Gorgon Univ

Washington• One of the first plant transformation Washington

University• Career at CibaNovartisSyngenta

Pre-transcriptional gene regulation by methylation of DNA

and acetylation of histones

Special proteins (e.g. chromomethylases) maintain

methylation patterns

Switching a gene on and off through DNA methylation and histone modification

Arabidopsis MET1 Cytosine Methyltransferase

Mutants Kankel et al. 2003. 163 (3):1109 Genetics

Plants mutant for MET1 show late-flowering phenotypes

Histone acetyl transferases and chromatin remodeling allows promoters to be accessible to

RNAPII

Histone tails are modified and can be studied easily

Figure 6.9

Some post-translational modifications

• Phosphorylation• Biotinylation• Glycosylation• Acetylation• Alkylation• Methylation• Glutamylation• Glycylation• Isoprenylation

• Lipoylation• Phosphopantetheinyl

ation• Sulfation• Selenation• C-terminal amidation

Phosphorylation is important for intracellular signalling

http://www.scq.ubc.ca/wp-content/uploads/2006/07/phosphocascades.gif

Protein glycosylation in the ER

The central dogma revisited

•The order of the DNA template or coding strand is 3’ to 5’

•This determines the order of the mRNA strand (5’ to 3’) because DNA template is complementary to the mRNA strand.

Figure 6.5

Eukaryotic gene structure and transcription of DNA into mRNA

TF

TF

AAAAARNA

polymerase II

TFTF

TF

Manipulating gene expression

• Can be done at several levels– Promoters, enhancers, transcription factors– Post-transcriptional– Translational – Methylation

• Biotechnology typically manipulates promoter

• Post-transcriptional gene silencing (RNAi) increasingly important