BIOL351 Exam 2 (ch. 6b, 7)

Transcription

• RNA synthesis off DNA template

• yields mRNA, tRNA, rRNA, regulatory RNAs

RNA

• ribose instead of deoxyribose

• uracil instead of thymine

• single-stranded

• fold into secondary structure that influences function

Transcription in Bacteria

• RNA synthesis off DNA template

• catalyzed by RNA polymerase

• phosphodiester bonds between ribonucleotides

• only one DNA strand transcribed

• no priming needed

• transcription terminators mark end of transcription

• highly regulated process allowing transcription at differing frequencies

RNA Polymerase

• 5 different subunits forming RNA polymerase holoenzyme complex

  • sigma not as tighly bound, easily dissociates to yield RNA polymerase core enzyme

  • core enzyme synthesizes RNA

  • sigma recognizes initiation sites on DNA called promoters to start transcription

Promoters

• specific DNA sequences

• recognized by sigma 70

• sequences vary but there are two highly conserved regions

  • Pribnow Box

    • -10 region, TATAAT

  • TTGACA (-35 region)

• alternative sigma factors recognize different consensus sequences

Strong Promoters

promoters conforming most closely to consensus sequences more effective in binding RNA polymerase

Sigma Factor 70

• RpoD

• TTGACA

• major housekeeping sigma factor for normal growth

Sigma Factor 54

• RpoN

• TTGGCACA

• nitrogen assimilation

Sigma Factor 38

• RpoS

• CCGGCG

• stationary phase

• oxidative and osmotic stress

Sigma Factor 32

• RpoH

• TNTCNCCTTGAA

• heat shock response

Sigma Factor 28

• FliA

• TAAA

• for genes involved in flagella synthesis

Sigma Factor 24

• RpoE

• GAACTT

• response to misfolded proteins in periplasm

Sigma Factor 19

• Fecl

• AAGGAAAAT

• iron transport

Transcriptional Units

• DNA segments transcribed into 1 RNA molecule bounded by initiation and termination sites

• can result in 1 or 2+ genes (cotranscribed genes)

• most genes encode proteins, but some encode untranslated RNAs (rRNA, tRNA)

Polycistronic RNA

• from transcribed operons

• contains multiple open reading frames that encode amino acids

Termination of Transcription

• governed by specific DNA sequences

• GC-rich sequence:

  • RNA forms stem-loop by intra-strand base pairing, RNA polymerase pauses, DNA-RNA dissociate

• Rho-dependent:

  • Rho protein recognizes DNA sequence and releases RNA polymerase from DNA

Transcription in Archaea and Eukarya

• similar to each other

• more complex than in bacteria

• Archaea have one RNA polymerase that resembles eukaryotic polymerase II

• Eukaryotes have 3 polymerases

• RNA processing of primary transcript required to form mature RNAs for translation

Eukaryotic Genes

• have coding and noncoding regions

• exons - coding

• introns - noncoding

  • found in tRNA and rRNA genes of Archaea

Protein Composition

• polymers of amino acids

  • compounds containing both amino and carboxylic acid groups liked to an alpha carbon

• amino acids linked by peptide bonds through carboxyl carbon and amino nitrogen

• polypeptide: many amino acids linked

• proteins consist of one or more polypeptides

• side chain - R group bonded to an alpha carbon

Protein Structure

• many unique proteins with different biochemical properties

• primary structure

  • linear array of amino acids

• secondary structure

  • from hydrogen bonding

• tertiary structure

  • 3D shape of polypeptide from hydrophobic interactions

• quaternary structure:

  • number and types of polypeptides (subunits) that make a protein

Transfer RNA (tRNA)

• carry amino acids to translation machinery

• each has an anticodon

  • 3 bases that recognize codon

• tRNA and cognate (correct) amino acid brought together by aminoacyl-tRNA synthetases

• single stranded

• extensive secondary structure

• contain bases modified post-transcription

Recognition and Activation of tRNA

• requires specific contacts for recognition

• amino acid activated by ATP to form aminoacyl-AMP

• amino acyl-AMP is attached to CCA stem of tRNA

• aminoacyl-tRNA complex leaves synthetase and will be bound ribosome

• codon recognition occurs by specific base pairing with complementary anticodon sequence on tRNA

  • some tRNAs recognize more than one codon

  • wobble: irregular base pairing allowed at 3rd position on tRNA

Degenerate Code

• multiple codons encode a single amino acid

• lacks one-to-one correspondence

Start Codon

• translation begins with AUG

• encodes N-formylmethionine in bacteria

• methionine in Archaea and Eukarya

Reading Frame

triplet code requires translation to begin at the correct nucleotide

Shine-Dalgarno Sequence

• ribosome-binding site (RBS)

• ensures proper reading frame in Bacteria

Stop Codons

• nonsense codons

• terminate translation

• UAA, UAG, UGA

• sometimes unusual amino acids selenosysteine and pyrrolysine can be encoded by stop codons

Open Reading Frame

• ORF

• AUG followed by a number of codons and a stop codon

Mechanism of Protein Synthesis

• initiation, elongation, termination

• uses mRNA, tRNA, ribosomes

• need guanosine triphosphate (GTP) for energy

Ribosomes

• large complexes of proteins and RNA where proteins are biosynthesized

• composed of two subunits

  • 30S and 50S to form 70S ribosome

  • 30S contains 16S rRNA + 21 proteins

  • 50S contains 5S + 23S rRNA + 31 proteins

Initiation Complex

• 30S subunit, mRNA, formylmethionine tRNA, initiation factors form

• 50S subunit added = 70S ribosome

• ribosome binding site

  • 3-9 nucleotides toward 5’ end of mRNA, complementary to sequences on 3’ end of 16s rRNA

• base pairing holds ribosome-mRNA complex in frame

Translation Initiation

• 2 ribosomal subunits + formylmethionine tRNA + initiation factors assemble mRNA

• begins at AUG

Translation Elongation

• amino acids brought to the ribosome and added to the growing polypeptide

• occurs in the A (acceptor) and P (peptide) sites of ribosome

• mRNA threads through ribosome

• tRNAs interact at A and P sites of 550S

  • A site: incoming charged tRNA first attaches; loading assisted by elongation factor EF-Tu

  • P site: growing polypeptide chain is attached to prior tRNA

• growing polypeptide moves to tRNA at the A site as new peptide bond is formed

Translocation Translation

• tRNA holding peptide transferred to P site

• ribosome advances 1 codon along mRNA per translocation

• amino acid free tRNA pushed to E and released from ribosome

Polysomes

a complex formed by multiple ribosomes simultaneously translating a single mRNA

Termination

• occurs at stop codon

• release factors (RFs) recognize stop codon and cleave polypeptide from tRNA

• ribosomal subunits dissociate

• subunits free to form new initiation complex and repeat process

Chaperones

• catalyze macromolecular folding events

• in E. coli

• DnaK, DnaJ

  • ATP-dependent enzymes that slow polypeptide folding

• GroEL, GroES

  • fold partially folded proteins

• can refold partially denatured proteins

• type of heat shock protein

  • attempt to refold partially denatured proteins for reuse before proteases destroy them

• help assemble cofactor-containing enzymes

Cold Shock Proteins

• prevent secondary structure formation in RNA or refold cold-sensitive proteins

Protein Secretion

• some proteins must be transported outside cytoplasmic membrane into periplasm or outer membrane

• typically requires ATP, GTP, or proton motive force

Translocases

• transport proteins into or through bacterial and archaeal membranes

• sec exports unfolded proteins and inserts integral membrane proteins

Signal Sequence

• at N-term of protein to be transported

• 15-20 residues, typically containing positively charged amino acids followed by hydrophobic residues and ending with polar residues

• signals export to translocase

• prevents protein from completely folding

• early export steps may begin before protein in completely made

Bacterial/Archaeal vs. Eukaryote Gene Arrangement

• bacteria/archaea gene arrangement

  • lacks introns

  • can be arranged in operons

    • two or more genes transcribed under control of promoter region located upstream where RNA polymerase initiates transcription

• bacterial/archaeal promoters characterized by distinct nucleotide sequences recognized and bound by DNA-binding proteins

  • allows RNA polymerase to bind, transcription to occur

Interaction of Proteins with Nucleic Acids

• small molecules influence the binding of regulatory proteins to DNA

  • turns transcription on/off

• most DNA-binding proteins interact with DNA in a sequence-specific manner

• specificity provided by interactions between amino acid side chains and chemical groups on the bases and sugar-phosphate backbone of DNA

• major groove of DNA is the main site of protein binding

Inverted Repeats

• nucleotide sequence followed downstream by inverted complement

• inverted repeats frequently are specific binding sites for regulatory proteins

Homodimeric

• two identical polypeptides

• DNA-binding proteins are often homodimeric

• each polypeptide has a domain that binds to one inverted repeat

Transcription Factors

• proteins that control the rate of transcription by binding to specific DNA

  • activator protein

  • repressor protein

Activator Protein

• turns on transcription

• binds DNA and recruits RNA polymerase or sigma factor to promoter region

Repressor Protein

• turns off expression

• binds operator region of DNA downstream of promoter

Effectors

• small molecules that control binding of activators and repressors

• typically cell metabolites (substrates, products) or structural analogs

Allosteric Proteins

• conformation altered when effector molecule binds

• since transcription factors are allosteric, conformational change determines whether transcription factor can bind DNA

• inducers

  • turn on transcription

• corepressors

  • turn off transcription

Enzyme Repression

• preventing synthesis of an enzyme unless product is absent from culture medium

• excess of product decreases enzyme synthesis

• specific effect

  • synthesis of all other enzymes continues normally

• widespread as control for production of amino acid and nucleotide precursors

• final product of a biosynthetic pathway is the corepressor effector molecule

• affects biosynthetic/anabolic enzymes

Enzyme Induction

• opposite of repression

• production of an enzyme in response to presence of substrate

• typically affects degradative/catabolic enzymes

• ensures enzymes are synthesized only when needed

Mechanisms of Repression and Depression

• repressors turn off transcription

• corepressors only bind DNA in presence of its effector

• arginine becomes corepressor when plentiful

  • binds arginine repressor (ArgR)

  • results in allosteric change and operator binding

  • since arg mRNA is polycistronic, all peptides encoded are repressed

• some repressors bind in absence of effector

  • lacl binds to operator, blocks transcription

    • if lacl effector present, combines with repressor, causing allosteric change that prevents lacl from binding DNA

    • transcription can proceed

    • corepressor = inducer

    • allolactose and IPTF are lac inducers

• repressor’s role is inhibitory so its called negative control

• genes are not turned on and off completely

  • often very low level of basal transcription when fully repressed

Mechanisms of Activation

• some operons transcribed only if activator protein first bound to DNA

• promoter sequences are poor matches to consensus promoter sequences, thus only weakly bind RNA polymerase

• positive control

  • regulator protein facilitates transcription

• activator proteins help RNA polymerase recognize promoter

  • may bend DNA structure

  • may interact directly with RNA polymerase

• activator proteins bind specifically to activator-binding site

Operons versus Regulons

• genes for maltose are spread out over the chromosome in several operons

  • each operon has an activator-binding site

  • maltose activator protein controls transcription of more than one operon

    • multiple operons/genes controlled by the same regulatory protein are called a regulon

• regulons exist for negatively controlled systems

Signal Transduction

• prokaryotes regulate cellular metabolism in response to environmental fluctuations

  • external signal may be detected by a sensor and transmitted to regulatory machinery

Two Component Regulatory Systems

• most signal transduction systems

• ex.

  • sensor kinase

    • cytoplasmic membrane

    • detects environmental signal and autophosphorylates at specific histidine residue (histidine kinase)

  • response regulator

    • in cytoplasm

    • DNA-binding protein that regulates transcription

    • receives phosphate from sensor kinase

• also has a feedback loop

  • terminates response or goes on forever

  • uses phosphatase (removes phosphate from response regulator)

Chemotaxis

• moving toward attractants

• moving away from repellents

• respond to temporal gradients (change in concentration over time)

• bacteria use modified two-component systems to sense temporal changes in attractants or repellents and regulate flagellar rotation

  • thus regulate activity of preexisting proteins instead of modifying transcription of genes

Response to Signal

• depends upon signal cascade of multiple proteins

• methyl-accepting chemotaxis proteins

  • sensory proteins that sense attractants and repellents and interact with cytoplasmic sensor kinases

    • chemoreceptors (clusters of thousands of MCPs)

  • E. coli has 4 transmembrane chemoreceptors (each contains 5 different MCPs)

  • MCP binding of attract or repellent triggers interactions with CheA (sensor kinase) and CheW

    • increase in repellent increases autophosphorylation of CheA

    • phosphate transferred to CheY (response regulator) that controls flagellar rotation

Controlling Flagellar Rotation

• CheY governs direction of rotation

• run

  • swim smoothly

  • counterclockwise

• tumble

  • move randomly

  • clockwise

• When MCPs bind repellent/release attractant, CheY-P (phosphorylated) interacts with flagellar motor to induce clockwise rotation and tumbling

• when MCPs bind attractant/release repellent, unphosphorylated CheY does not bind to flagellar motor

  • results in CCW rotation and running

Adaptation

• stop responding and reset sensory system

  • feedback loop resets the system

    • relies on CheB (response regulator)

    • involved methylation of MCPs

      • methylation - stops response to attracts/increases response to repellents

      • unmethylated - respond strongly to attractants/insensitive to repellents

    • CheR methylates and CheB-P demethylates

Phototaxis

• movement toward light

• light sensor replaces MCPs

• sensors interact with cytoplasmic Che proteins

  • leads to runs/tumbles

Aerotaxis

• movement toward oxygen

• redox protein monitors oxygen level

• sensors interact with cytoplasmic Che proteins

  • leads to runs/tumbles

Cell-to-Cell Signaling

• prokaryotes can communicate through production of small extracellular molecules

  • small peptides or nonpeptide organics

  • accumulation leads to coordinated group behaviors

    • ex. biofilm formation

Quorum Sensing

• regulatory mechanism by which Bacteria and some Archaea assess their population density

• ensures sufficient # of cells are present before initiating activities that requires a certain cell density to be effective

• each species produces a specific autoinducer signaling molecule

  • diffuses freely across the cell envelope

  • reaches high concentrations inside cell only if many cells are nearby and making the same autoinducer

  • binds to specific activator protein or sensor kinase triggering transcription of specific genes

• first discovered as mechanism regulating light production in bacteria including A. fischeri

Acyl Homoserine Lactone (AHL)

• first autoinducer to be identified

• several types found in Gram Negatives

Autoinducer 2 (AI-2)

• a common autoinducer among many Gram Negative species, allowing interspecies communication

Short Peptides

• used as autoinducers by Gram Positives and Archaea

E. Coli Virulence Factors

• pathogenesis

• ex. E. coli

  • Shiga toxin-producing strain

  • produces AHL AI-3 that induces virulence genes

    • activates 2 transcriptional activators

      • activating motility, toxin secretion, production of lesion forming proteins

S. Aureus Virulence Factors

• secretes small peptides that damage host cells or interfere with host’s immune system

• under control of autoinducing peptide (AIP)

  • activates several proteins that lead to production of virulence proteins

• quorum-sensing disruptors could be potential drugs for dispersing biofilms and preventing virulence gene expression

Global Control Systems

• regulate transcription of many different genes in more than one regulon

• may include activators, repressors, signal molecules, 2-component regulatory systems, regulatory RNA, alternative sigma factors as components

Lac Operon

• lactose operon and maltose regulon respond to global controls

• catabolite repression

  • controls use of carbon sources if more than one present

    • glucose always used first

  • synthesis of unrelated catabolic enzymes is repressed if glucose is present in growth medium

  • also called “glucose effect”

  • ensure that the “best” carbon and energy source is used first

Catabolite Repression

• better energy source consumed first, growth stops

• after lag, growth resumes with 2nd energy source

Cyclic AMP Receptor Protein (CRP)

• activator protein

• form of activation

• allosteric and binds to DNA only if it has first bound Cyclic AMP

  • regulatory nucleotide derived from adenosine

  • synthesized by adenylate cyclase

Lac Gene Transcription

• for lac genes to be transcribed

  • cyclic AMP level must be high enough for CRP to bind to CRP-binding site (positive control)

  • lactose or another inducer must be present to prevent lactose repressor (Lacl) binding (negative control)

Stress Survival Pathways

• improve chances of survival under harsh conditions

Stringent Response

• widely distributed regulatory mechanism to survive nutrient deprivation, environmental stress, and antibiotics

• shuts down macromolecule synthesis and activates pathways to improve survivability

E. coli Stringent Response

• if shifted down from amino acid excess to limitation, rRNA and tRNA synthesis stops and no new ribosomes produced

• protein and DNA synthesis stop, new amino acids biosynthesized

• later, rRNA synthesis and new ribosome production begin again at a slower rate

• triggered by 2 regulatory nucleotides/armones

  • guanosine tetraphosphate ppGpp

  • guanosine pentaphosphate pppGpp

General Stress Response

• the RpoS Regulon

• entering stationary phase of growth is a response to nutrient limitation and stressors

• controlled by alternative sigma factor RpoS

  • stationary phase sigma factor

• RpoS regulon includes 400+ genes

  • nutrient limitation

  • resistance to DNA damage

  • biofilm formation

  • responses to osmotic, oxidative, and acid stress

Heat Shock Response

• global control mechanism to protect cells from protein denaturation resulting from heat, high solvent levels, osmotic stress, UV light

• temperature and stress can generate large amounts of inactive proteins that need to be refolded or degraded

• HSP counteract damage of denatured proteins and help cell recover from stress

  • Hsp100

    • proteases that degrade denatured/aggregated proteins

  • Hsp90

  • Hsp70

    • DnaK

  • Hsp60

    • GroEL

  • Hsp10

    • GroES

The Alternative Sigma Factor RpoH

• heat shock response controlled by alternative sigma factor RpoH

  • controls heat shock protein expression

  • degraded within 1-2 minutes of synthesis

  • when heat shock occurs, RpoH degredation inhibited, level increases, increasing transcription of operons who promoters are recognized

  • RpoH degredation depends on level of DnaK, which inactivates RpoH

  • RpoH mRNA basepairs with itself, regulating translation

• Also heat shock response in Archaea and eukaryotes

Noncoding RNA (ncRNA)

• RNA that is not translated to protein

• includes rRNA, tRNA, signal recognition particle RNA

• also includes small RNA (sRNA)

Small RNA (sRNA)

• 40-400 nucleotides that regulate gene expression in prokaryotes and eukaryotes

• basepair directly to other RNAS (typically mRNA)

• binding modulates rate of target mRNA translation because double-stranded RNA cannot be translated

Riboswitches

• metabolite binds directly to mRNA

• results in control at either transcriptional or translational level

• aptamer region (switch)

  • recognition domain that binds small molecules

    • 2 alternative secondary structures

      • bound with/without small molecule

• alternation between forms depends on presence/absence of small molecule, which controls expression platform (secondary structure of downstream RNA)

  • secondary structure controls whether ribosome binds and begins translation

  • or controls whether RNA polymerase transcribes mRNA

• bound metabolite usually product of pathway whose enzymes are encoded by the mRNA that carry the riboswitches

Evolution of Riboswitches

• found in some Bacteria, Archaea, few plants, and fungi

• believed to be remnants of RNA world

  • before cells, DNA, and protein were present

  • catalytic RNAs were only self-replicating “life”

  • could have been metabolic control mechanism

Attenuation

• transcriptional control that functions by premature termination of mRNA synthesis

• control exerted after the initiation of transcription, but before its completion

  • # of completed transcripts reduced but # initiated is not

Leader

• first part of mRNA structure

• can fold into 2 alternative secondary structures either allowing synthesis or causing premature termination

• ex. tryptophan operon in E. coli

• not found in eukaryotes because transcription and translation are separated (organelles)

Attenuation in the Tryptophan Operon

• trp operon contains structural genes for 5 proteins + promoter and reguatory sequences at beginning

• has more than one type of regulation

• transcription of whole operon controlled by repressor and corepressor (tryptophan)

• leader sequence containing tandem tryptophan codons (attenuator) encodes leader peptide

• if plenty of trp, plenty of charged trp tRNAs, and leader peptide synthesized, resulting in termination of transcription of the rest of the trp operon

• if little trp, leader peptide not synthesized, and rest of operon transcribed

Mechanism of Attenuation

• transcription and translation are simultaneous

• transcription attenuated because new mRNA folds into a stem-loop that inhibits RNA polymerase

• if plenty of trp, leader sequence forms stem-loop

  • pauses transcription and triggers termination

• if little trp, ribosome pauses at trp codon because of trp tRNA shortage

  • new, different stem-loop forms

  • prevents termination stem-loop from forming

• rate of transcription is influenced by rate of translation

Feedback Inhibition

• mechanism for temporarily turning off the reactions in a biosynthetic pathway

  • end product of the pathway binds to an early (often first) enzyme in pathway

    • results in the shutting down of the pathway since no intermediates are generated

  • reversible reaction

    • once levels of end product are limiting, pathway functions in reverse

  • inhibited enzyme has 2 binding sites

    • active (substrate-binding)

    • allosteric (end product binds)

      • binding at allosteric site changes conformation, prevents substrate binding