REVIEW FOR MIDTERM:

SYNTHETIC BIOLOGY
TEXAS A & M INTERNATIONAL UNIVERSITY

BIOL 4471/5471 101 FL17

Synthetic Biology-WIN

Judge M Garza & Sabrina Garza

09-22-2017

Dr. Sebastian Schmidl

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CONTENTS
1 Molecular Information Flow ........................................................................................................................5
1.1 Molecular Biology and Genetic Elements ...........................................................................................5
1.1.1 DNA and Genetic Information Flow ...........................................................................................5
1.1.2 DNA Structure .............................................................................................................................5
1.1.3 Size of DNA .................................................................................................................................5
1.1.4 Shape of DNA ..............................................................................................................................5
1.1.5 Genetic Elements .........................................................................................................................5
1.1.6 Plasmids .......................................................................................................................................5
1.1.7 R(esistance) Plasmids ..................................................................................................................6
1.1.8 Transposable Elements ................................................................................................................6
1.1.9 Central Dogma .............................................................................................................................6
1.2 Copying the Genetic Blueprint: DNA Replication ..............................................................................6
1.2.1 DNA Replication..........................................................................................................................6
1.2.2 DNA Polymerase .........................................................................................................................6
1.2.3 Primer Synthesis ..........................................................................................................................7
1.2.4 DNA Replication: Initiation .........................................................................................................7
1.2.5 DNA Replication: Elongation ......................................................................................................7
1.2.6 Connecting DNA Fragments on the Lagging Strand ...................................................................7
1.2.7 DNA Replication: Termination ....................................................................................................7
1.2.8 Replisome Complex .....................................................................................................................7
1.2.9 DNA Polymerase: Proofreading ..................................................................................................8
1.3 RNA Synthesis: Transcription .............................................................................................................8
1.3.1 Transcription ................................................................................................................................8
1.3.2 RNA Polymerases ........................................................................................................................8
1.3.3 Process of RNA Synthesis ...........................................................................................................8
1.3.4 Bacterial Promoters ......................................................................................................................8
1.3.5 Bacterial Terminators ...................................................................................................................8
1.3.6 Transcription in Bacteria ..............................................................................................................8
1.3.7 Bacterial Transcription Inhibitors ................................................................................................9
1.4 Protein Synthesis: Translation .............................................................................................................9
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1.4.1 amino acids, polypeptides, and proteins ......................................................................................9

1.4.2 amino acid structure .....................................................................................................................9
1.4.3 protein structure .........................................................................................................................10
1.4.4 translation and the genetic code .................................................................................................10
1.4.5 Messenger rna ............................................................................................................................11
1.4.6 Transfer rna ................................................................................................................................12
1.5 Protein Processing, Folding, Degradation, & Secretion ....................................................................12
1.5.1 Protein processing ......................................................................................................................12
1.5.2 protein folding and chaperones ..................................................................................................13
1.5.3 other functions of chaperons ......................................................................................................13
1.5.4 Protein degradation ....................................................................................................................13
1.5.5 bacterial clp proteases ................................................................................................................14
1.5.6 protein export .............................................................................................................................14
1.5.7 sec system: protein export into the cytoplasmic membrane .......................................................14
1.5.8 sec system: protein eport to the periplasm .................................................................................15
1.5.9 tat system ...................................................................................................................................15
1.5.10 protein secretion: gram-negative bacteria ..................................................................................15
1.5.11 protein secretion: gram positive bacteria ...................................................................................16
2 Microbial Regulatory Systems ...................................................................................................................16
2.1 DNA-Binding Proteins and Transcriptional Regulation ....................................................................16
2.1.1 major modes of regulation .........................................................................................................16
2.1.2 monitoring gene expression .......................................................................................................16
2.1.3 dna binding proteins ...................................................................................................................17
2.1.4 structure of dna binding proteins................................................................................................17
2.1.5 dna regulatory sequences ...........................................................................................................18
2.1.6 negative control: repression and induction ................................................................................18
2.1.7 negative control: regulaton of arginine operon ..........................................................................18
2.1.8 negative control: regulation of lactose operon ...........................................................................18
2.1.9 positive control: activation .........................................................................................................18
2.1.10 global control and the lac operon ...............................................................................................18
2.1.11 lactose permease ........................................................................................................................18
2.1.12 transcriptional induction of the lac operon.................................................................................18
2.2 Sensing and Signal Transduction .......................................................................................................19
2.2.1 sensing and signal transduction..................................................................................................19
2.2.2 two-component regulatory systems............................................................................................19
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2.2.3 envz-ompr two-component regulatory system ...........................................................................19

2.2.4 quorum sensing ..........................................................................................................................19
2.2.5 Microbial bioluminescense ........................................................................................................19
2.2.6 Interspecies communication .......................................................................................................19
2.2.7 quorum sensing regulation of virulence factors .........................................................................19
2.2.8 Phosphate (pho) regulon ............................................................................................................19
2.3 Integrated Control Circuits.................................................................................................................19
2.3.1 Stringet response ........................................................................................................................19
2.3.2 Biofilms and cyclic-di-gmp signaling ........................................................................................20
2.3.3 General stress response: rpos regulon ........................................................................................20
2.3.4 Heat shock response ...................................................................................................................20
2.3.5 Inactivation of sigma factors ......................................................................................................20
2.3.6 Other Global control systems .....................................................................................................20
2.4 RNA-Based Regulation......................................................................................................................21
2.4.1 Regulatory rnas ..........................................................................................................................21
2.4.2 Types of regulatory rnas ............................................................................................................21
2.4.3 Riboswitches ..............................................................................................................................21
2.5 Regulation of Enzyme Activity..........................................................................................................21
2.5.1 feedback inhibition.....................................................................................................................21
2.5.2 post-translational regulation .......................................................................................................22
2.5.3 regulation of Nitrogen metabolism ............................................................................................22
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1 MOLECULAR INFORMATION FLOW

1.1 MOLECULAR BIOLOGY AND GENETIC ELEMENTS

1.1.1 DNA AND GENETIC INFORMATION FLOW

Nucleic Acids- Contain information via nucleotides

1.1.2 DNA STRUCTURE

Properties of the double helix
o Backbone of DNA chain consists of alternating Phosphates and the pentose sugar
deoxyribose
o Phosphodiester bonds connect 3-carbon of one sugar to 5 -carbon of the adjacent sugar
o DNA is double stranded held together by hydrogen bonds
o Complementary base pairs (AT) (CG)
o Antiparallel
o DNA interactions in major groove

1.1.3 SIZE OF DNA

Expressed in base pairs (bp)

1.1.4 SHAPE OF DNA

Relaxed DNA- # of turns predicted by bp
Supercoiled DNA twisted upon itself to save space
o Negative supercoiling- under twisted helix with larger gaps
o Positive supercoiling Over twisted with smaller gaps
Topoisomerase- insert and remove super coils
DNA Gyrase- introduces supercoils into DNA by double strand breaks

1.1.5 GENETIC ELEMENTS

Chromosome main genetic element with housekeeping genes
Virus genomes
Plasmids
Organelle genome
Transposable elements

1.1.6 PLASMIDS
Double-stranded DNA that replicate separately from the chromosome
o Usually circular
o Ranges in size from one kbp to more than one Mbp
May confer a selective growth advantage Under certain conditions
Variable Abundance
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1.1.7 R(ESISTANCE) PLASMIDS

Confer resistance to antibiotics or other growth inhibitors
Important for horizontal gene transfer (Conjugation)

1.1.8 TRANSPOSABLE ELEMENTS

Discrete segments of DNA that can move as a unit from one location to another on the same or a different
DNA molecule
Move by transposition
o Transposase recognizes, cuts, and ligates DNA
o Insertion of a transposable element generates a duplicated target sequence
o Two types of transposition
Conservative - cut and paste
Replicative - copy and paste
Two main types of transposable elements in bacteria are
o insertion sequences (IS)
carries gene encoding for transposase (only protein encoded)
have inverted repeats at ends
smaller than transposons
o transposons (Tn)
carries gene encoding for transposase
have inverted repeats at ends
moves DNA between inverted repeats
Many genes inside

1.1.9 CENTRAL DOGMA

Replication TranscriptionTranslation

1.2 COPYING THE GENETIC BLUEPRINT: DNA REPLICATION

1.2.1 DNA REPLICATION

DNA replication is semiconservative
o One parental (template) strand and one new strand
Precursor of each nucleotide is a deoxynucleotide 5-triphosphate (dNTP)
Steps
1. Initiation- recognition of origin of replication and binding to it
2. Elongation- unwinding DNA through the enzyme DNA Helicase, synthesizing and
connecting lagging strands.
3. Termination completion of copying and proofreading

1.2.2 DNA POLYMERASE

Catalyze polymerization of deoxynucleotides
Forms new strands of DNA using primer: template junctions as a guide
Five different DNA polymerases in E.coli
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o DNA Pol III primary enzyme replicating chromosomal DNA

o DNA Pol I plays lesser role in DNA replication (Lagging strand)

1.2.3 PRIMER SYNTHESIS

DNA polymerase requires a primer for DNA elongation which is made by DNA primase
o Binds to open DNA and makes a short RNA copy (RNA primer)
o Important to start on a DNA strand
o RNA primer is found on both DNA strands

1.2.4 DNA REPLICATION: INITIATION

DNA synthesis begins at the origin of replication in prokaryotes
o DnaA: recognizing initiator protein and separates double helix
o DnaB: loads onto DNA & unwinds
o DnaC: loader protein
DnaB unwinds the DNA along replication fork

1.2.5 DNA REPLICATION: ELONGATION

DNA helicase (DnaB) unwinds DNA
Replication is always from the 5 to 3 ends
leading strand synthesized 5 to 3
lagging strand synthesized in short 5 to 3 fragments (Okasaki Fragments)
DNA synthesis is bidirectional due to circular chromosome.
o Shape called theta structure

1.2.6 CONNECTING DNA FRAGMENTS ON THE LAGGING STRAND

Synthesis continues on lagging strand until it reaches previously synthesized DNA
DNA Pol I removes RNA primer and replaces with DNA
o 5 to 3 exonuclease activity
DNA ligase seals nicks in DNA

1.2.7 DNA REPLICATION: TERMINATION

Termination occurs at the terminus of replication when the chromosome has been copied.
o Contains DNA sequences call Ter sites
Replication termination protein Tus interacts with Terminus region
o Blocks progress of the replication forks
Separation of the linked chromosome due to the enzyme topoisomerase IV

1.2.8 REPLISOME COMPLEX

Replisome complex of multiple proteins involved in replication
DNA is pulled through the replisome.
Contains key replication proteins.
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1.2.9 DNA POLYMERASE: PROOFREADING

DNA replication is accurate.
Proofreading is essential to minimize mutations
DNA Pol I & III contain a 3 to 5 exonuclease activity to remove improperly paired bases.

1.3 RNA SYNTHESIS: TRANSCRIPTION

1.3.1 TRANSCRIPTION
Process by which DNA is transcribed into RNA by the enzyme RNA polymerase (RNAP)
o RNAP uses DNA as template.
o RNAP can start a trend without having the primer
o No proofreading
o Chain growth is 5 to 3
o Only 1 strand transcribed

1.3.2 RNA POLYMERASES

Core enzyme has 5 subunits
Eukaryotes 3 RNAPs (each is responsible for a specific subset of RNA
o More complex than bacterial RNAP
Archaea contain only one RNAP
o Resembles euk. RNAP II

1.3.3 PROCESS OF RNA SYNTHESIS

RNAP core enzyme associates with sigma factor to form RNAP holoenzyme.
o Recognizes promoters (site of initiation of transcription)
Transcription stops at the transcription terminators.
Transcription involves smaller units of DNA.
o Allows cell to transcribe different genes at different rates.

1.3.4 BACTERIAL PROMOTERS

Sigma factors recognize 2 regions of the promoter
o Pribnow box located 10 base pairs before the start of transcription
o -35 regions located 35 base pairs upstream from start site

1.3.5 BACTERIAL TERMINATORS

Termination of RNA synthesis is controlled by specific DNA sequences
o Intrinsic termination transcription stops without additional factors.
o Rho-dependent termination Rho protein binds to specific DNA sequences and cause RNA
polymerase to pause, which then releases RNA & RNAP.

1.3.6 TRANSCRIPTION IN BACTERIA

Transcriptional unit DNA segments transcribed into a RNA molecule
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o Bounded by initiation and termination sites

Some RNAs are not translated (rRNA & tRNA)
o both are very stable
o 3 types of rRNA : 16S, 23S, 5S
mRNAs have short half-lives (degraded by ribonucleases)
Bacteria has genes clustered together
o Allows expression of multiple genes to be coordinated
Operon group of related genes that are cotranscribed
Polycistronic mRNA mRNA encoding a group of cotranscribed genes
o Contains multiple open reading frames, or the part that actually encodes amino acids

1.3.7 BACTERIAL TRANSCRIPTION INHIBITORS

Rifamycins macrocyclic antibiotics produced by Amycolatopsis rifamycinica
o Inhibit bacterial RNA polymerase by binding to the beta subunit (RpoB)
o Rifampin resistance due to mutations in rpoB or Arr-catalyzed ADP-ribosylation
Actinomycin D -antibiotic produced by soil bacteria of the order Actinomycete
o Has phenoxazone ring system
o Blocks transcription elongation by mimicking a DNA base

1.4 PROTEIN SYNTHESIS: TRANSLATION

1.4.1 AMINO ACIDS, POLYPEPTIDES, AND PROTEINS

Proteins play major role in cell functions
o Catalytic proteins, Structural proteins, Regulatory proteins
Amino acids are linked through peptide bonds to form a polypeptide
Proteins consist of one or more polypeptides

1.4.2 AMINO ACID STRUCTURE

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1.4.3 PROTEIN STRUCTURE

Primary structure linear array of
amino acids
Secondary structure formation of
alpha helix or beta sheet due to
hydrogen bonding
Tertiary structure three-
dimensional shape of polypeptide
Quaternary structure number and
types of polypeptides that make a
protein

1.4.4 TRANSLATION AND THE GENETIC CODE

Translation- synthesis of proteins from RNA
o Requires all 3 types of RNA
Genetic code made up of codons
o Codons triplet of nucleic acid bases that encode for a single amino acid
o 64 possible codons
o Specific codons for starting (AUG) and for stopping (UAA, UAG, UGA)
AUG anticodon binds to methionine
UAA, UAG, & UGA anticodons do not bind to any A.A.
o Degenerate- multiple codons encode for a single A.A.
o Wobble flexibility in the third nucleic acid. Causes irregularity, most of the time its has no
effect since the code is degenerate
o Codon bias- multiple codons for the same amino acid are not used equally
Varies between organelles and organisms
Correlates with tRNA availability and concentration
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1.4.5 MESSENGER RNA

mRNA- RNA that is transcribed from DNA to be read by ribosomes for translation
Reading Frame- triplet code requires translation to begin at the correct nucleotide.

Shine-Dalgarno sequence- (ribosome binding site) recruits the ribosome through complementary base
pairing
o Ensures proper reading frame
Open reading frame (ORF) Start codon, codons in between, stop codon
o Start codon (AUG) encodes for N-formylmethionine (fMet) in bacteria and methionine in
archaea and eukaryotes. Begins translation.
o Stop codon (UAA, UAG, UGA) terminate translation
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1.4.6 TRANSFER RNA

tRNA- attaches to a specific amino acid for
translation
single stranded
cloverleaf shape
amino acid binds to CCA 3 terminal group of the
acceptor end
anticodon - Three bases on tRNA that recognize
three complementary bases on mRNA

tRNA and amino acid coupled by aminoacyl-

tRNA

1.5 PROTEIN PROCESSING, FOLDING, DEGRADATION, &

SECRETION

1.5.1 PROTEIN PROCESSING

Complete proteins released from ribosome have fMet at the N-terminus
Protein processing involves 2 enzymes:
o Formylmethionine deformylase cuts off N-formyl group leaving just methionine
o Methionine aminopeptidase removes the entire amino acid
Addition of acetyl groups or AMP can change protein function
o Examples of modified bacterial enzymes:
Ribosomal proteins acetylation
Glutamine synthetase adenylyation
Isocitrate dehydrogenase phosphorylation
Proteolytic cleavages can activate or inactivate a protein
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1.5.2 PROTEIN FOLDING AND CHAPERONES

Chaperones catalyze macromolecular folding events

o Assist in folding & are not incorporated into
protein
o Highly conserved
Major chaperones in E.coli
o DnaK/DnaJ -ATP dependent enzyme that
slows down polypeptide folding
o GroEL/GroES folds partially folded
proteins

1.5.3 OTHER FUNCTIONS OF CHAPERONS

Also known as heat shock proteins
o Synthesis is accelerated due to stress by heat
o Refold partially denatured proteins for reuse
Cold shock proteins:
o Produced at cold temperatures
o Function as RNA chaperones

Some chaperones (as above) help assemble cofactor-containing enzymes

1.5.4 PROTEIN DEGRADATION

Many proteins contain degrons degradation signals.
o Dictate stability
o Includes: short amino acids sequences, structural motifs, & exposed amino acids located
anywhere in the protein
N-terminal rule degron that states the n-terminal amino acid correlates with its stability
o Short half-life (2 mins-) - begins with leucine, phenylalanine, tryptophan or tyrosine
o Longer half-life aspartic acid, glutamic acid, or cysteine
Degradation of short-lived is facilitated by ClpS
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1.5.5 BACTERIAL CLP PROTEASES

Highly conserved
Present in all bacteria
Similar to structure of eukaryotic proteasomes
Protease complex has a barrel-shaped structure
o Two stacked rings of proteolytic subunits are sandwiched between 2 rings or single-capped by 1
ring of ATPase-active chaperon subunits

1.5.6 PROTEIN EXPORT

Translocases proteins that transport specific proteins through and into prokaryotic membranes
Secretory (sec) system exports unfolded proteins and inserts integral membrane proteins into
cytoplasmic membrane
o Proteins contain a signal sequence
Found at N-terminus
Prevents complete folding
o SecYEG assist in exporting to the periplasm or insertion into the membrane
Twin-arginine translocase (Tat) system transports folded proteins through the cytoplasmic membrane
o Contains amino acid consensus motif S/T-R-R-X-F-L-K in N-terminal sequence

1.5.7 SEC SYSTEM: PROTEIN EXPORT INTO THE CYTOPLASMIC MEMBRANE

Recognition of signal sequence by signal recognition particle (SRP)
o Halts translation process of integral cell membrane proteins
Route to membrane depends on the protein inserted
o Directed by protein FtsY
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1.5.8 SEC SYSTEM: PROTEIN EPORT TO THE PERIPLASM

Interaction of trigger factor with newly synthesized proteins keep them in a loosely folded conformation
The Sec pathway translocates the periplasmic proteins
o SecB, SecA, & SecYEG
o ATP as energy
Periplasmic signal peptidases cut off signal sequences after translocation

1.5.9 TAT SYSTEM

Twin-arginine translocase (Tat) system transports folded proteins through the cytoplasmic membrane
into the periplasm
Contain highly conserved twin-argine leader motif
Composed of three essential membrane proteins, TatA, TatB, & TatC
Powered by proton motive force
Important for proteins that contain small cofactors such as iron-sulfer or redox-coupled proteins

1.5.10 PROTEIN SECRETION: GRAM-NEGATIVE BACTERIA

6 different secretion systems
Transport proteins or small molecules (effectors) into the outer membrane or completely out of the cell
Forms channel through membrane
Facilitate symbiosis, biofilm formation, enzyme secretion, dna transfer, antibiotic release, & protein
delivery
Type II and V
o Two step translocase- depend on Sec or Tat system
o Do not require ATP or proton motive force
o Type II includes a secretion pore in the outer membrane for type II system proteins
o Type V simplest system that is called auto transporter
Types I, III, IV, and VI secretion systems:
One-step translocases Form channels through both cytoplasmic and outer membranes
Do not depend on Sec or Tat
Usually require ATP
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o Type I Composed of cytoplasmic membrane transporter, outer membrane pore, and membrane
fusion protein
o Type III Injects toxins into eukaryotic host cells
o Type IV Most common and normally transfer DNA through conjugation
o Type VI Cytoplasmic injector

1.5.11 PROTEIN SECRETION: GRAM POSITIVE BACTERIA

Clustering of secretion systems and accessory factors at the cytoplasmic membrane in an anionic
phospholipid microdomain called the ExPortal
Located near the cell septum
Linked to peptidoglycan synthesis
Proteins in the ExPortal include:
HtrA Assists in pili formation and covalent attachment of proteins to the cell wall
Sortase Aids maturation of secreted proteins
Sec system components
Chaperones

2 MICROBIAL REGULATORY SYSTEMS

2.1 DNA-BINDING PROTEINS AND TRANSCRIPTIONAL
REGULATION

2.1.1 MAJOR MODES OF REGULATION

Gene expression transcription of a gene into mRNA followed by translation of mRNA into a protein
o Constitutive - Proteins and RNA molecules needed at the same level
Microbial genomes encode proteins that are not always needed
o Helps conserve energy
2 approaches to regulations
o Controlling the activity of preexisting proteins
Post-translational regulation
Very rapid process
o Controlling the amount of a protein
Regulates level of transcription
Regulates translation
Slower

2.1.2 MONITORING GENE EXPRESSION

Reporter genes encode for an easy-to-detect product
o Can fuse to other genes
o Can fuse to regulatory elemen9ts
o Ex. Green fluorescent protein (GFP)
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2.1.3 DNA BINDING PROTEINS

Transcription regulation requires proteins that bind to DNA
o Affinity with DNA is often influenced by small molecules
Most DNA binding proteins interact with DNA in a sequence specific manner
o Major grove of DNA main site of protein binding
o Inverted repeats binding site for regulatory proteins
DNA binding proteins are often homodimeric
o Made of two identical polypeptide subunits which consist of domain regions
Protein dimers interacts with inverted repeats

Many possible outcomes after protein binds to DNA

o Catalyze a specific reaction (ex. Transcription by RNA polymerase)
o Negative regulation binding can block transcription
o Positive regulation binding can activate transcription

2.1.4 STRUCTURE OF DNA BINDING PROTEINS

Classes of protein domains critical for proper binding to DNA
o Helix-turn-helix
Composed of 2 -helices connected by a short sequence of amino acids which form the
turn
First helix recognition helix
Second helix stabilizing helix
o Zinc finger
Contains cysteine and histidine residue that binds a zinc ion
Euk. Regulatory proteins use for DNA binding
o Leucine zipper
Regularly spaced leucine residues
Function hold two recognition helices in the correct orientation (scissor-like
configuration)
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2.1.5 DNA REGULATORY SEQUENCES

DNA binding proteins can bind at low levels to any sequences
o Scan for high affinity binding
Promoters can recruit the service of a regulatory protein through evolution

2.1.6 NEGATIVE CONTROL: REPRESSION AND INDUCTION

Negative control regulatory mechanism that stops transcription
o Repression prevents the synthesis of an enzyme in response to sufficient amounts of a product
Affects anabolic enzyme (arginine biosynthesis)
o Induction production of an enzyme in response to a substrate
Typically affects catabolic enzymes (lac operon)

2.1.7 NEGATIVE CONTROL: REGULATON OF ARGININE OPERON

Acts as a corepressor by binding to the allosteric repressor protein (ArgR)

2.1.8 NEGATIVE CONTROL: REGULATION OF LACTOSE OPERON

2.1.9 POSITIVE CONTROL: ACTIVATION

2.1.10 GLOBAL CONTROL AND THE LAC OPERON

2.1.11 LACTOSE PERMEASE

2.1.12 TRANSCRIPTIONAL INDUCTION OF THE LAC OPERON

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2.2 SENSING AND SIGNAL TRANSDUCTION

2.2.1 SENSING AND SIGNAL TRANSDUCTION

2.2.2 TWO-COMPONENT REGULATORY SYSTEMS

2.2.3 ENVZ-OMPR TWO-COMPONENT REGULATORY SYSTEM

2.2.4 QUORUM SENSING

2.2.5 MICROBIAL BIOLUMINESCENSE

2.2.6 INTERSPECIES COMMUNICATION

2.2.7 QUORUM SENSING REGULATION OF VIRULENCE FACTORS

2.2.8 PHOSPHATE (PHO) REGULON

2.3 INTEGRATED CONTROL CIRCUITS

2.3.1 STRINGET RESPONSE

Stringent Response Mechanism used to survive nutrient deprivation, environmental stress, and
antibiotics exposure. It shuts down macromolecule synthesis and or activates stress survival pathways.
o Environment/habitat determines response
o When nutrients are lowr:
rRNA and tRNA synthesis temporarily stops
Protein and DNA synthesis ceases as well
Activation of amino acid biosynthesis
rRNA synthesis and new ribosome production begins again at slower rate after adaptation
phase
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Stringent response is triggered by

o (p)ppGpp- mixture of Mixture of the alarmones guanosine tetraphosphate (ppGpp) and guanosine
pentaphosphate (pppGpp)
(p)ppGpp have global control effects by activating stress response pathways as well as
biosynthetic amino acid operons
Gram-negative bacteria stop rRNA and tRNA synthesis by binding to RNA
polymerase and preventing transcription of genes for these RNAs
Gram-positive bacteria Interfere with initiating ribonucleotides for
transcription
o RelA Enzymes synthesizing alarmones using ATP as phosphate donor
Associated with the SOS subunit of the ribosome
Activated by a signal from the ribosome during amino acid limitation
o Gpp Protein that converts pppGpp to ppGpp
o SpoT Protein that either makes or degrades (p)ppGpp depending of environmental condition
Example 1:
o Bacteria moves from your intestines with plenty of nutrients to the toilet when you take a fat shit.
The limit of nutrients in the toilet water initiates the production of ppGpp which causes the
bacteria to slow down their cell division
Example 2:
o Caulobacter crescentus is a bacterium that normally lives in freshwater that are low in nutrients.
Their stringent response is triggered by low carbon and ammonia levels rather than amino acid
availability. This causes production of ppGpp which increases the motile cells (swimmers) to be
developed. Now that these mother fuckers can swim, they can go to areas that are richer in
nutrients.

2.3.2 BIOFILMS AND CYCLIC-DI-GMP SIGNALING

2.3.3 GENERAL STRESS RESPONSE: RPOS REGULON

2.3.4 HEAT SHOCK RESPONSE

2.3.5 INACTIVATION OF SIGMA FACTORS

2.3.6 OTHER GLOBAL CONTROL SYSTEMS

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Global control systems Regulate many genes comprising more than one regulon. Includes activators, repressors,
signal molecules, two-component systems, alternative sigma () factors, and regulatory RNAs

2.4 RNA-BASED REGULATION

2.4.1 REGULATORY RNAS

2.4.2 TYPES OF REGULATORY RNAS

2.4.3 RIBOSWITCHES

2.5 REGULATION OF ENZYME ACTIVITY

2.5.1 FEEDBACK INHIBITION

Feedback Inhibition mechanism for temporarily
turning off the reactions in a biosynthetic pathway
End product binds and inhibits activity of an early
enzyme
o Reversible reaction based on non-covalent
interactions
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Inhibited enzyme is an allosteric enzyme with two binding

sites:
o Active site substrate binding
o Allosteric site end product binding

2.5.2 POST-TRANSLATIONAL REGULATION

2.5.3 REGULATION OF NITROGEN METABOLISM