Ch 18.1-4, Campbell 9th edition Gene regulation Ch 18.1-4, Campbell 9th edition

Lac operon – inducible operon 18.1 Gene Regulation in Prokaryotes: Lac operon – inducible operon Normally, the repressor IS bound to the operator, so lac operon is OFF. In the "induced" state, the lac repressor is NOT bound to the operator site.

Trp operon - repressible Trp operon is normally ON When tryptophan is present it binds to the repressor, which activates it. The activated repressor will bind to the operator, “repressing” the operon

Positive Gene Regulation Positive activation of lac operon Bacteria uses carbohydrates for energy – glucose vs. lactose How is this signaled to the bacteria?

Positive activation is through the CAP protein, which is activated by cAMP. cAMP accumulates when glucose is scarce.

18.2 Gene Regulation in Eukaryotes Gene is expressed when it makes a protein Expression regulated at various levels: Chromatin structure Transcription factors* Alternative splicing Non-coding RNAs that degrade other mRNAs

Chromatin packing

Chromatin structure Histone acetylation- acetyl groups are added to histones loosens chromatin structure – promotes transcription Deacetylation – acetyl groups removed, reduces transcription Methylation – methyl groups added to certain bases in DNA Reduces transcription in some species In genomic imprinting, regulates expression of maternal or paternal alleles of certain genes

Regulation of Transcription: Typical Eukaryotic Gene Organization Control elements, segments of noncoding DNA, are associated with eukaryotic genes. Control elements act as binding sites for transcription factors that help regulate transcription Control elements and the transcription factors that bind them allow for precise control of gene regulation

Eukaryotic gene & transcript Enhancer (distal control elements) DNA Upstream Promoter Proximal control elements Transcription start site Exon Intron Poly-A signal sequence Transcription termination region Downstream Poly-A signal Transcription Cleaved 3 end of primary transcript 5 Primary RNA transcript (pre-mRNA) Intron RNA RNA processing mRNA Coding segment 5 Cap 5 UTR Start codon Stop codon 3 UTR 3 Poly-A tail P G AAA  AAA

Transcription factors – general ones are required for the RNA polymerase binding. They bind first to the DNA, and then recruit the RNA polymerase. Specific transcription factors bind with control elements for regulation Enhancers – groups of control elements upstream of a gene, have binding sites for specific transcription factors

Activators - a protein that binds to an enhancer and stimulates transcription of a gene have two domains, one that binds DNA and a second that activates transcription Repressors - transcription factors that inhibit expression of a particular gene by a variety of methods

MyoD – a specific transcription factor that acts as an activator MyoD is a master regulatory gene DNA Activation domain DNA-binding domain

Gene Switches http://www.hhmi.org/biointeractive/gene-switch

Activators DNA Enhancer Distal control element Promoter Gene TATA box General transcription factors DNA- bending protein Group of mediator proteins RNA polymerase II RNA synthesis Transcription initiation complex

Model for Transcription Initiation 1. Transcriptional activators bind to DNA & recruit chromatin remodeling complexes and histone acetyltransferases 2. These open up the chromatin to expose promoter & regulatory sequences 3. Transcriptional factors bind to enhancers 4. DNA bending protein protein brings activators, mediator proteins, and general transcirption factors together to form transcription initiation complex on promoter

Regulation of Eukaryotic DNA Transcription http://www.hhmi.org/biointeractive/regulation-eukaryotic-dna-transcription

Coordinately controlled genes in eukaryotes Genes coding for enzymes of a metabolic pathway are often scattered over different chromosomes Coordinate gene expression depends on simultaneous expression of the genes Chemical signalling is often used for coordinate gene expression – i.e. hormones

Alternate Gene Splicing Post transcriptional regulation through RNA processing Different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

1 2 3 4 5 Exons DNA Troponin T gene Primary RNA transcript RNA splicing or mRNA 1 2 3 4 5

mRNA degradation Lifespan of mRNA in cytoplasm affects protein synthesis mRNA in eukaryotes lasts longer than prokaryotic mRNA

Translation Initiation of translation can be blocked by proteins that bind to parts of mRNA

Protein processing & degradation Post-translational protein processing includes cleavage, and addition of functional groups Proteasomes are giant protein complexes that bind protein molecules and degrade them

18.3 Non coding mRNAs A large part of the genome is made up of DNA that is transcribed into non-coding mRNAs (ncRNA) These can affect translation and chromatin expression

RNA Important in many cellular machines: Ribosome rRNA Spliceosome snRNA Telomerase telomerase RNA

Interference with Translation MicroRNAs (miRNA) are single-stranded RNA molecules that can bind to mRNA Small Interfering RNA (siRNA) act similarly to miRNAs, but have a longer, double stranded precursor They can degrade mRNA or block its translation This is called RNA interference - RNAi

(a) Primary miRNA transcript Hairpin miRNA Hydrogen bond Dicer miRNA- protein complex mRNA degraded Translation blocked (b) Generation and function of miRNAs 5 3

microRNAs A novel class of ncRNA gene Products are ~22 nt RNAs Precursors are 70-100 nt hairpins Gene regulation by pairing to mRNA Unknown before 2001 Forms RISC – RNA inducing silencing complex

Small Interfering RNAs - siRNA RNA interference (RNAi) – when double stranded RNA injected into the cell, it turned off expression of gene with same sequence as the RNA siRNAs are the cause of this RNAi Similar to miRNA, but formation is different Many siRNAs are formed from a longer, double stranded RNA molecule Some siRNAs can bind back to chromatin and cause changes in the chromatin

siRNA

Chromatin & ncRNA In some yeasts, siRNAs can play role in heterochromatin forming, and block parts of chromosome Small ncRNAs can induce heterochromatin, which blocks parts of chromosome, blocking transposons

RNAi (~5 min)] http://www.youtube.com/watch?v=cK-OGB1_ELE

18.4 Differential gene expression leads to different cell types in multicellular organism One fertilized egg can give rise to many different cell types Differential gene expression results from genes being regulated differently in each cell type Materials in the egg can set up gene regulation that is carried out as cells divide

Cell development Zygote cell – totipotent – has potential to develop into a complete organism Cell determination – cell has committed to a final fate, it is unable to change at this point Cell differentiation – cell produces tissue-specific proteins, cell has clear cut identity Morphogenesis – organization of cells into tissues & organs

Cytoplasmic Determinants Based on uneven distribution of cytoplasmic determinants in egg The cytoplasm has RNA & proteins that were encoded by the mother’s DNA When cell divides, the two cells have different amounts of the determinants, which can determine the cell’s fate

(a) Cytoplasmic determinants in the egg Unfertilized egg Sperm Fertilization Zygote (fertilized egg) Mitotic cell division Two-celled embryo Nucleus Molecules of two different cytoplasmic determinants

Induction signals and cell differentiation Environment around the cell, especially signals from nearby embryonic cells influence development of cells The changes in gene expression lead into observable cellular changes In the process called induction, signal molecules from embryonic cells cause transcriptional changes in nearby target cells Interactions between cells induce differentiation of specialized cell types

Signal Induction - types Signals from one group of cells influence another group of cells Diffusion: signal diffuses from distance to receptor– i.e. hormone, or other signal molecule - receptor can transmit signal through second messengers in signal transduction pathway

Signal Induction - types Direct contact – neighboring cells Gap junction – cytoplasm of 2 cells is connected

Muscle cell determination

Pattern formation What controls the body plan of an organism? How do organs get in the right place? 2 general models: Morphogen gradient Sequential induction

Sequential induction Differentiation due to production & release of a series of chemical signals

Morphogen gradient A diffusible chemical signal, or morphogen, is produced. The concentration is higher closer to the source, and lower farther away from the source. The fate of the cell depends on its exposure to the different threshold levels.

Drosophila- model organism Lewis studied development by looking at mutants with bizarre developmental defects, and through this discovered homeotic genes Homeotic genes specify the identity of body segments Mutations in these genes lead to structures in the wrong place

In a fruit fly, for example, Hox genes lay out the various main body segments—the head, thorax, and abdomen. Here we see a representation of a fruit fly embryo viewed from the side, with its anterior end to the left and with various Hox genes shown in different colors. Each Hox gene, such as the blue Ultrabithorax or Ubx gene, is expressed in different areas, or domains, along the anterior-to-posterior axis.

Drosophila development Cytoplasmic determinants in egg establish axes of drosophila body Bicoid mRNA from mother is translated into the Bicoid protein in the Drosophila zygote Bicoid is transcription factor that turns on genes in different levels

a. Bicoid concentration & 4 genes affected b. concentration gradient of Bicoid in zygote– more at right c. concentration gradient in embryo after several divisions d. hunchback protein – green, kruppel protein - orange

Eric Wieschaus – Bicoid gradient (3:28) http://www.youtube.com/watch?v=pAoK-KOUTZM Bicoid animation (2:15) http://www.youtube.com/watch?v=uaedzlrnBGY

Bonnie Bassler – Quorum sensing & gene expression http://media.hhmi.org/hl/09Lect2.html?start=32:28&end=39:40 http://www.hhmi.org/biointeractive/molecular-cascade-bacterial-quorum-sensing

miRNA slides from: sgj@sanger.ac.uk http://www.sanger.ac.uk/Software/Rfam/ rfam@sanger.ac.uk