CHAPTER 15: Regulation of Gene Expression in 15.2 Lactose Metabolism in E.

coli Is Regulated
Bacteria by an Inducible System

15.1 Bacteria Regulate Gene Expression in Lactose metabolism

Response to Environmental Conditions ● its enzymes are inducible
● lactose: inducer
Inducible Enzymes
● Synthesis stimulated by presence of Operons
specific substrates or environ conds ● cluster genes expressing enzymes for a
● Regulated expression by inducer related function under control of single
molecules promoter
● ex. lac operon
cis-acting site
Constitutive Enzymes ● DNA sequence that regulates the
● Synthesized continuously regardness of expression of nearby genes on the same
presence/absence of substrates or environ DNA molecules
conds ● cis-acting regulatory regions
● ex. glycolysis enzymes ○ promoters, operators, enhancers

Repressible trans-acting factors

● Synthesis of enzymes inhibited by ● molecules (proteins/RNA) regulate gene
presence of specific end products of a expression but not connected to DNA they
metabolic pathway regulate
● Downregulated when conc. of end prod ● ex. repressor proteins
exceeds cell's needs
● ex. trp operon lactose (lac) operon
● controls expression of genes involved in
Negative Control lactose metabolism
● Regulation of gene expression by
inhibitory factors Structural genes
○ repressor proteins ● found on the operon region
○ regulatory RNA ● lacZ: B-galactosidase
● ex. lac repressor ● lacY: lactose permease
● lacA: thiogalactoside transacetylase
Positive Control
● Regulation of gene expression by lac- mutants
stimulatory factors ● [Link] mutants that cant metabolize
○ activator proteins lactose
○ regulatory RNA
● ex. CAP lacOc mutants
● [Link] with constitutive expression of lac
operon

[Discovery of Regulatory Mutations]

Gratuitous inducers
● Isopropyl B-D-1-thiogalactopyranoside
(IPTG): analog of lactose that induce lac
operon in absence of glucose
Constitutive mutations 15.3 Catabolite-Activating Protein (CAP) Exerts
● Mutations resulting from continuous Positive Control over the lac Operon
expression of gene
● Even if there are inducers or repressors Catabolite-activating protein (CAP)
● ex. constitutive mutation in lac repressor ● Binds to sequences near certain
gene promoters
● Enhances transcription of associated
Repressor gene genes
● Encodes protein that bind to operator
region of operon and inhibit transcription Catabolite repression
● ex. I gene of lac operon ● Regulatory mechanism in bacteria where
○ mutation = consti lac operon presence of preferred carbon sources
inhibits expression of genes involved in
Operator region (O region) using alternative carbon sources
● DNA sequence in promoter region where
repressor binds to Glucose absent
● mutation = consti lac operon ● cAMP levels rise and CAP is activated
● cAMP-CAP binds to CAP-binding site and
[Operon Model: Negative Control] enhance transcription of genes that uses
alt carb sources
Operon model
● Regulatory region Glucose present
○ promoter and operator sequences ● cAMP levels decrease
○ Structural genes ● CAP cannot bind
■ transcribed together as one ● RNA polymerase seldom binds and
mRNA transcription is inhibited

leader sequence cyclic adenosine monophosphate

● before the start codon of the first ● accumulates in cells when glucose levels
structural in an operon are low
● adenyl cyclase synthesizes cAMP from ATP
Repressor molecule
● allosteric: changes conformation when
bound to inducer (lactose)

When lactose is absent

● lac repressor binds to operator region
● physically blocking RNA polymerase

When lactose is present in

● lactose binds to lac repressor
● lac repressor is altered and cant bind to
operator
● transcription of structural genes

[Isolation of Repressor]

Equilibrium dialysis
● Technique used to study binding of
molecules (proteins) to small molecules
(inducers or substrates)
15.4 The Tryptophan (trp) Operon in [Link] is a [Riboswitches]
Repressible Gene System
Riboswitches
tryptophan synthase ● In 5’ untranslated regions of mRNA (UTRs)
● catalyzes the final steps of biosynthesis of that bind to small molecule ligands
trp ○ metabolites

corepressor Aptamer
● small molecule that binds to repressor ● ligand binding domain of riboswitch
protein
● they bind to operator region and inhibit Expression platform
transcription ● regulatory domain of riboswitch that
changes in response to ligand
Five contiguous structural genes
● trpE, D, C, B, A Default vs Antiterminator conformation
● Default: terminator hairpin forms
tryptophan absent ● Antiterminator: makes antiterminator
● trp repressor cant bind to corepressor trp hairpin and transcription conts
● trp operon is transcribed
[Small noncoding RNAs play regulatory roles in
ENOUGH tryptophan present Bacteria]
● trp binds to trp repressor
● trp is not transcribed small noncoding RNAs (sRNAs)
● base-pairs with target mRNAs or proteins
15.5 RNA Plays Diverse Roles in Regulating ● modulates stability, translation, or activity
Gene Expression in Bacteria
15.6 CRISPR-Cas Is An Adaptive Immune
[Attenuation] System in Bacteria

Attenuation Innate immunity

● regulatory mechanism that control gene ● First line of defense against pathogens
expression by premature termination of ● present from birth
transcription
● forms 2ndary in mRNA transcription Adaptive immunity
○ termination/continuation ● Acquired immune response developed
○ depende ra daw from exposure to pathogens
● antigens bind to lymphocytes ->
Antiterminator hairpin antibodies -> immunological memory
● 2ndary structure in mRNA
● prevents premature termination Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR)
Terminator hairpin ● bacterial immune system that provides
● 2ndary structure acquired immunity
● signals premature termination ● derived from previous exposures to
foreign genetic elements
Attenuator
● regulatory sequence in leader region CRISPR-associated (cas) genes
● sequences for the 2ndary structures! ● adjacent to CRISPR loci
● important to adaptive immunity
CRISPR-Cas mechanism steps
1. spacer acquisition
● CRISPR system incorporates short
DNA sequences from invading
genetic elements
● memory of past infections
2. CRISPR RNA (crRNA) biogenesis
● CRISPR array transcription and
processing to mature crRNA
● guides CRISPR-Cas to target
specific NA sequences
3. target interference
● CRISPR-Cas uses crRNA to
recognize and bind to invading
nucleic acids
● Cas proteins cleave target
DNA/RNA and boom no more
infection
Cas9
● CRISPR-associated endonuclease enzyme
for CRISPR-Cas9 genome editing
CHAPTER 16: Regulation of Gene Expression in movement and interaction of chromatin
Eukaryotes fibers and nuclear proteins

Multicellular eukaryotic organisms Open Chromatin Conformation

● cell types had diverse morpho and funcs 1. Variant and unstable histone
● some genes only expressed under certain 2. Histone acetylation by histone
conditions or times
acetyltransferase (HAT)
a. acetylation makes histone less
Importance of regulation
● misregulation can lead to: developmental
positive and reduces affinity to
defects, disease, over expression negative Phosphate group of DNA
backbone
Chromatin remodeling 3. Chromatin remodeling complexes
- chromatin has to be open to RNA polymerase to begin a. uses ATP hydrolysis to move and
transcription but having it in a closed conformation rearrange nucleosomes
inhibits access to the DNA.
Closed Chromatin Conformation
16.1 Organization of the Eukaryotic Cell 1. Normal barrier histone composition
Facilitates Gene Regulation at Several Levels 2. Histone deacetylases

DNA Methylation
PROKARYOTE EUKARYOTE ● methylation of cytosine at 5’ =>
5-methylcytosine
free unwound DNA form chromatin with ● Occurs more on cytosine of GG doublets in
histones DNA

everything in transcription in
cytoplasm nucleus; CpG Islands
translation in ● Methylable CpG are concentrated in such
cytoplasm islands
● In or near promoter regions. 70% of
no splicing splicing of pre-mRNA
human genes have CpG sa promoter
translated mRNAs have long
immediately and half-lives; transported, DNA Methylation and Gene Regulation
degraded rapidly localized, and ● Repress transcription by inhibiting binding
translated in specific of transcription factors to DNA
subcellular ● Recruitment of repressive chromatin
destinations
remodeling complexes to gene-regulatory
regions
16.2 Eukaryotic Gene Expression is Influenced
by Chromatin Modification

Nucleosomes
● Repeating DNA-histone complex

Chromosome territory
● spatial organization of chromosomes
within the nucleus

Interchromatin compartments
● regions in nucleus that separate chromatin
domains for dynamic environment for
16.3 Eukaryotic Transcription Initiation 16.4 Eukaryotic Transcription Initiation is
Requires Specific Cis-Acting Sites REgulated by Transcription Factors Bind to
Cis-Acting SItes
cis-acting DNA elements
● located on the same chromosome as the Transcription factors
gene they regulate ● binds to promoters, enhancers, and
silencers
Promoter
● Region of DNA recognized and bound by General transcription factors (GTFs)
basic transcription machinery ● required for basic process of transcription
● Specify site/sites at which transcription initiation
begins (transcription start site)
Activators
Core promoters ● increase levels of transcription initiation
● Minimum part of promoter ● physically interacts with HAT
● Needed for accurate initiation of ○ if bound to enhancers, recruit more
transcription by RNAP II HAT para more open

Proximal promoter elements Repressors

● ~250 nucleotides upstream of transcription ● reduce transcription levels
start site
Human metallothionein 2A (MT2A) gene
Focused core promoters ● example of how transc of one gene can be
● specify transcription initiation at a single regulated by the interplay of multiple
specific start site promoter and enhancer elements and
transc factors that bind them
Dispersed core promoters ● expressed at low or basal level in all cells
● for weak transcription start sites 50-100 ● transcribed at high levels when cells are
nucleotide region exposed to heavy metals or
glucocorticoids
Core-promoter elements
● Short nucleotide sequences that are 16.5 Activators and Repressors Interact with
bound by specific regulatory factors General Transcription Factors and Affect
Chromatin Structure
Initiator element (Inr)
● Encompasses transcription start site pre-initiation complex (PIC)
● critical in the initiation of transc; RNAP II
Enhancers + GTFs
● located on either side of a gene; position
is critical coactivators
● cis regulatory elements ● proteins that serve as a bridge between
● increase the rate of transcription activators and promoter-bound GTFs
● inverted
enhanceosomes
Silencer ● large complexes of activators and
● cis; negative regulator of transcription coactivators that come together to direct
transcriptional activation

Activators and repressors act by enhancing or repressing the

formation of PI at the promoter, opening/closing chromatin,
and/or relocating genes to specific nuclear sites
16.6 Regulation of Alternative Splicing 16.7 Gene Expression Is Regulated by mRNA
Determines Which RNA Spliceforms of a Gene Stability and Degradation
Are Translated
steady-state level of mRNA
Spliceforms ● total amount at any point in time
● spliced pre-mRNA that may or may not ● function of rate at which gene is
have exons transcribed and rate at which mRNA is
degraded
Alternative splicing ● determines amt of mRNA available for
● enables single gene to encode more than translation
one variant of its protein product
(isoforms) Exoribonucleases
○ may have different functions or ● degrade RNA via removal of terminal
active sites nucleotides
● Exosome complex: destroys mRNA in 3’-5’
Cassette exons manner
● may be excluded from mature mRNA
● joining 3’-end of upstream exon to 5'-end Deadenylation-dependent decay
of downstream exon ● most euka mRNAs degrade thru
deadenylases
Alternative splice site
● 25% of alternative splicing events in Decapping enzymes
animals ● recruited by shortened poly-A tails
● Usage are important regulatory events ● remove 5’ cap and allow CRN1 to destroy
mRNA in 5’ to 3’
Intron retention
● introns are included in mature mRNAs Deadenylation-independent decay
● translated to produce novel isoforms ● begins at 5’ cap
● but ma degrade dayun :((( ● endoribonucleases

Mutually exclusive exons mRNA surveillance response

● swapping of protein domains encoded by ● mRNA with premature stop codon triggers
different exons this
● premature stop codons are caused by
Alternative promoters nonsense mutations
● more than one site where transcription
may be initiated nonsense-mediated decay
● efficiently eliminates mRNAs with
Alternative polyadenylation premature stop codons
● polyadenylation directs termination and
Modulation of mRNA stability and decay can eliminate
addition of poly-A tail
aberrant mRNAs and regulate gene expression

Proteome
● # of proteins an organism can make

RNA-binding proteins
● regulates alternative splicing

Spliceopathies
● mutations that disrupt RNA splicing
16.8 Noncoding RNAs Play Diverse Roles in 16.9 mRNA Localization and Translation
Posttranscriptional Regulation Initiation Are Highly Regulated

noncoding RNAs (ncRNAs) Zip code

● serve as posttranscriptional regulators of ● cis-regulatory element
gene expressions ● found in lamellipodium that has Actin
mRNA
RNA Interference (RNAi) ● binding site for RNA-binding protein
● ncRNA guides posttranscriptional (RBP) called zip code binding protein 1
silencing of mRNAs (ZBP1)
● destroy mRNA or block their translation ○ binds to large subunit and blocks
● can be used in medicine to block translation initiation
translation of genes that cause excess
amount of products that lead to diseases Src
● phosphorylates ZBP1 para dli mag RNA
small noncoding RNAs (sncRNAs) binding
● small interfering RNAs (siRNAs)
○ Dicer cleaved from Some mRNAs are stored for later use and/or
double-stranded RNA localized to specific regions of the cell and then
○ associate with RNA-induced locally translated to create asymmetric protein
silencing complex (RISC) distribution within the cell
○ Endoribonuclease activity
○ For specific mRNA sequences for example, Actin mRNA is bound to ZBP1 and
● microRNAs (miRNAs) transported to the lamellipodium and once ZBP1
○ produced from transcription and is phosphorylated by Src, it is released and Actin
processing of miRNA genes can finally be translated
○ transcribed into primary miRNAs
(pri-miRNAs) 16.10 Post Translational Modifications Regulate
○ Drosha makes it into pre-miRNAs Protein Activity
○ miRNA-RISC binds to miRNA
response elements (MREs) Post-translational modifications
○ For variety of mRNAs ● regulation of gene products

long noncoding RNAs (lncRNAs) Regulation by Phosphorylation

● no start or stop codons => no protein ● Induces conformational changes; can be
● some bind to chromatin-regulating on or off based on effect on affinity
complexes ● Kinases: phosphorylation
● competing endogenous RNAs (ceRNAs) ● Phosphatases: dephosphorylation
○ soak up miRNAs since they also
have MREs Ubiquitin-Mediated Protein Degradation
○ decoys of miRNAs para dli pirmi ● Ubiquitination: ubiquitin is attached to
mag inhibit sa mRNA translation lysine of target protein
● Tags a protein for destruction
● Proteasome: recognizes
poly-ubiquitinated protein and cleaves it
● Ubiquitin ligases: recognize and bind
specific target proteins and catalyze the
progressive addition of ubiquitin residues