Gene Expression

Operons: The Basic Concept A cluster of functionally related genes can be under coordinated control by a single “on-off switch” The regulatory “switch” is a segment of DNA called an ___________ usually positioned within the promoter _________________is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control

The operon can be switched off by a protein ____________________ ______________________________by binding to the operator and blocking RNA polymerase

The repressor can be in an active or inactive form, depending on the presence of other molecules A______________________is a molecule that cooperates with a repressor protein to switch an operon off For example, E. coli can synthesize the amino acid tryptophan

Figure 18.3 trp operon Promoter Promoter Genes of operon DNA trpR trpE trpD trpC trpB trpA Operator Regulatory gene RNA polymerase Start codon Stop codon 3 mRNA 5 mRNA 5 E D C B A Protein Inactive repressor Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on DNA No RNA made Figure 18.3 The trp operon in E. coli: regulated synthesis of repressible enzymes. mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off

Polypeptide subunits that make up enzymes for tryptophan synthesis Figure 18.3a trp operon Promoter Promoter Genes of operon DNA trpR trpE trpD trpC trpB trpA Operator Regulatory gene RNA polymerase Start codon Stop codon 3 mRNA 5 mRNA 5 E D C B A Protein Inactive repressor Figure 18.3 The trp operon in E. coli: regulated synthesis of repressible enzymes. Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on

Tryptophan (corepressor) Figure 18.3b-1 DNA mRNA Protein Active repressor Figure 18.3 The trp operon in E. coli: regulated synthesis of repressible enzymes. Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off

Tryptophan (corepressor) Figure 18.3b-2 DNA No RNA made mRNA Protein Active repressor Figure 18.3 The trp operon in E. coli: regulated synthesis of repressible enzymes. Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off

Repressible and Inducible Operons: Two Types of Negative Gene Regulation _____________________________________________________________________________________________________________________ An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription © 2011 Pearson Education, Inc.

Figure 18.4 Regulatory gene Promoter Operator DNA DNA lacI lacZ No RNA made 3 mRNA RNA polymerase 5 Active repressor Protein (a) Lactose absent, repressor active, operon off lac operon DNA lacI lacZ lacY lacA RNA polymerase Figure 18.4 The lac operon in E. coli: regulated synthesis of inducible enzymes. 3 mRNA mRNA 5 5 Protein -Galactosidase Permease Transacetylase Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on

(a) Lactose absent, repressor active, operon off Figure 18.4a Regulatory gene Promoter Operator DNA DNA lacI lacZ No RNA made 3 mRNA RNA polymerase 5 Figure 18.4 The lac operon in E. coli: regulated synthesis of inducible enzymes. Active repressor Protein (a) Lactose absent, repressor active, operon off

Allolactose (inducer) Figure 18.4b lac operon DNA lacI lacZ lacY lacA RNA polymerase 3 mRNA mRNA 5 5 -Galactosidase Permease Transacetylase Protein Figure 18.4 The lac operon in E. coli: regulated synthesis of inducible enzymes. Inactive repressor Allolactose (inducer) (b) Lactose present, repressor inactive, operon on

________________________________________________________________________________________________________________________ Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

Positive Gene Regulation Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription

Evolution of gene regulation Eukaryotes multicellular evolved to maintain constant internal conditions while facing changing external conditions homeostasis regulate body as a whole ___________________________ long term processes __________________________ turn on & off large number of genes must coordinate the body as a whole rather than serve the needs of individual cells Specialization each cell of a multicellular eukaryote expresses only a small fraction of its genes Development different genes needed at different points in life cycle of an organism afterwards need to be turned off permanently Continually responding to organism’s needs homeostasis cells of multicellular organisms must continually turn certain genes on & off in response to signals from their external & internal environment

Points of control The control of gene expression can occur at any step in the pathway from gene to functional protein 1. packing/unpacking DNA 2. _______________ ______________

1. DNA packing How do you fit all that DNA into nucleus? DNA coiling & folding __________________________ nucleosomes chromatin fiber looped domains chromosome nucleosomes “beads on a string” 1st level of DNA packing histone proteins have high proportion of positively charged amino acids (arginine & lysine) bind tightly to negatively charged DNA from DNA double helix to condensed chromosome

Nucleosomes “Beads on a string” 1st level of DNA packing 8 histone molecules Nucleosomes “Beads on a string” 1st level of DNA packing __________________ 8 protein molecules positively charged amino acids _________________________ DNA packing movie

DNA packing as gene control Degree of packing of DNA regulates transcription tightly wrapped around histones ____________________ genes turned off H E

Histone acetylation ______________________________ loosely wrapped around histones enables transcription ________________________ conformational change in histone proteins transcription factors have easier access to genes

2. Transcription initiation Control regions on DNA ____________________________ nearby control sequence on DNA binding of RNA polymerase & transcription factors “________________________ _________________ distant control sequences on DNA binding of activator proteins “enhanced” rate (high level) of transcription

Model for Enhancer action Enhancer DNA sequences distant control sequences Activator proteins bind to enhancer sequence & stimulates transcription Silencer proteins bind to enhancer sequence & block gene transcription Much of molecular biology research is trying to understand this: the regulation of transcription. Silencer proteins are, in essence, blocking the positive effect of activator proteins, preventing high level of transcription. Turning on Gene movie

Transcription complex Activator Proteins • regulatory proteins bind to DNA at distant enhancer sites • increase the rate of transcription Enhancer Sites regulatory sites on DNA distant from gene Enhancer Activator Activator Activator Coactivator B F E RNA polymerase II A TFIID H Coding region T A T A Core promoter and initiation complex Initiation Complex at Promoter Site binding site of RNA polymerase

3. Post-transcriptional control ________________________ variable processing of exons creates a family of proteins

4. Control of translation _______________________________ regulatory proteins attach to 5' end of mRNA prevent attachment of ribosomal subunits & initiator tRNA _______________________ Control of translation movie

Gene Regulation 7 6 5 4 2 1 4 3 protein processing & degradation 1 & 2. transcription - DNA packing - transcription factors 3 & 4. post-transcription - mRNA processing - splicing - 5’ cap & poly-A tail - breakdown by siRNA 5. translation - block start of translation 6 & 7. post-translation - protein processing - protein degradation 5 4 initiation of translation mRNA processing 2 1 initiation of transcription mRNA protection mRNA splicing 4 3