Control of Gene Expression

Control of transcription (prokaryotic cells) An Operon (F. Jacob and J. Monod, 1961) is groups of genes that are regulated together. It includes structural genes coding for proteins that are functionally related, the operator and the promoter. A single mRNA transcript carries the coding information of an entire operon (polycistronic mRNA).

The Lactose Operon in E. coli I Promoter Operator Z Y A Terminator Repressor-binding site structural genes that code for enzymes which allow E. coli to use lactose a DNA sequence that marks the end of transcription Regulator gene encodes repressor protein a DNA sequence to which RNA polymerase binds to initiate transcription

The Lactose Operon (Negative control, repression) promoter operator Z, Y, A I terminator RNA Inactivated repressor RNA polymerase repressor lactose (inducer)

The Lactose Operon (positive control) promoter operator Z, Y, A terminator I RNA RNA polymerase CAP cAMP

The Lactose Operon Regulation positive negative (repressor protein) (CAP protein) Conditions: -lactose, +glucose The operon doesn’t work -lactose, -glucose The operon doesn’t work +lactose, +glucose Low level of transcription +lactose, -glucose High level of transcription

The Tryptophan Operon in E. coli Regulator gene promoter operator Structural genes terminator R P O At E D C B A T Attenuator Structural genes encode the enzymes required for the synthesis of trytophan

Negative control (trp operon) Regulator gene promoter operator terminator RNA Repressor protein activated repressor tryptophan (corepressor) RNA polymerase

Attenuation Attenuation is the early termination of transcription

The tryptophan operon Regulation attenuation negative (control of transcription by translation) (repressor protein)

Operons Repressible catabolic anabolic Inducible (trp operon) (lac operon) (trp operon) (lac operon)

Control of transcription (eukaryotic cells) Regulatory elements cis-acting trans-acting proteins promoters terminators enhancers silencers insulators LCR

Cis-acting elements Enhancers are DNA sequences to which specific transcription factors (activators) bind to increase the rate of transcription. Silencers are DNA sequences to which specific transcription factors (repressors) bind to decrease the rate of transcription. Properties of enhancers (silencers): can be upstream or downstream of the transcription initiation site may modulate from a distance of thousands of base pairs away from the initiation site. Loop formation

Insulators An insulator is a DNA sequence that blocks the interaction between enhancers and promoters. DNA promoter 1 insulator enhancer promoter 2 The enhancer activates transcription from the promoter 2 but not from the promoter 1.

LCR elements LCR (Locus Control Region) is a segment of DNA that controls transcription of an entire gene cluster, such as the beta-globin cluster in vertebrates. LCRs enhance the expression of linked genes in a tissue-specific manner.

DNA Methylation DNA methylation is a process where a methyl group is added to the cytosine in CpG-islands. CpG Islands are CpG rich regions located at 5’ ends of genes, usually in promoter regions. DNA methylation generally reduces transcription. It inhibits the binding of transcriptional activators.

Histone Modifications In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails (increasing of transcription) Methylation can condense chromatin (decreasing of transcription). Genes in highly compacted chromatin are generally not transcribed. Phosphorylation can activate transcription or condense chromatin.

The histone code hypothesis proposes that specific combinations of modifications, as well as the order in which they occur, help determine chromatin configuration and influence transcription (Jenuwein and Allis, 2001).

RNA Interference (Craig Mello and Andrew Fire, 1998, 2006) The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) RNA Interference is RNA induced post-transcriptional gene silencing (siRNA).

RNAi functions regulates expression of protein coding genes mediates resistance to exogenous and endogenous pathogenic nucleic acid (viruses, transposons) used experimentally to block gene expression

Control of translation Inhibition by antisense RNAs (prokaryotic cells). Antisense RNA is a single-stranded RNA (70-110 bases) that is complementary to a messenger RNA (mRNA) strand Riboswitches (prokaryotic cells) are regulatory segments of a messenger RNA molecule that selectively bind metabolites (small molecules) and control gene expression.

Control of translation Inhibition or activation of initiation factors (eIF4, eIF2 etc.) Inhibition by RNA binding proteins. Proteins that bind to specific sequences in the mRNA and prevent ribosomes from attaching can prevent translation of certain mRNA molecules.

Inhibitors of gene expression 1. Inhibitors of prokaryotic transcription: Actinomycin D binds to DNA and blocks transcription Rifampicin binds to RNA polymerase and blocks initiation of RNA synthesis Streptolydigin inhibits elongation of transcription by binding to RNA polymerase 2. Inhibitors of eukaryotic transcription: α-Amanitin inhibits RNA polymerase II and RNA polymerase III (at high doses). RNA polymerase I is insensitive

Inhibitors of gene expression 3. Inhibitors of prokaryotic translation: Streptomycin binds to the 30S subunit of the bacterial ribosome and blocks initiation Tetracycline inhibits the binding of aminoacyl-tRNA to the mRNA-ribosome complex. Chloramphenicol prevents protein chain elongation by inhibiting the peptidyl transferase activity of the bacterial ribosome. Puromycin is a structural analogue of the 3′ end of aminoacyl-transfer RNA. It binds to the A site of the ribosome and causes premature chain termination. 4. Inhibitors of eukaryotic translation: Diphtheria toxin and Cycloheximide block translocation of the ribosome.