Exam 1 Bio 411 PDF

Lecture 2 Growth Understand the factors involved in bacterial growth and how they influence the growth curve. ○ What are the 5 steps of the growth-complex process? Entrance of nutrients Conversion into energy and cell components Chr...

Lecture 2 Growth Understand the factors involved in bacterial growth and how they influence the growth curve. ○ What are the 5 steps of the growth-complex process? Entrance of nutrients Conversion into energy and cell components Chromosome replication Increase in size and mass Division ○ At any given time, the number of cells is represented by what equation? b = 1x2^n What does n represent? ○ Number of doublings or generations ○ What 5 factors influence the growth curve? Nutrients What do all organisms need nutrients wise? ○ Carbon, energy, nitrogen, phosphorus, oxygen, etc. What is the difference between an autotroph (lithotroph) and a heterotroph (organotroph)? ○ Autotrophs - get carbon from CO2 and inorganic compounds ○ Heterotrophs - get carbon from glucose or organic compounds What is the difference between chemo and photo energy sources? ○ Chemo - inorganic/organic compounds ○ Photo - light The richer the medium… ○ The bigger the growth rate Nutritional composition of the medium will determine what 2 things? ○ Specific growth rate ○ Total growth and stationary phase density The level of the stationary phase is dependent on? The medium Cells with oxygen typically? ○ Grow faster Temperature What happens at low temps? ○ Membrane gelling - cell membrane becomes rigid or “gel like” When this happens, the rigid state inhibits transport processes (moving nutrients in and out)  ○ What happens at high temps? Protein denaturation - collapse of the membrane, thermal lysis (cells destroyed) ○ What happens to the rate of reactions as temp increases? They get faster pH Water activity You need water for bio reactions (water = polar, water loving) Oxygen 5 classifications ○ Obligate aerobes - need oxygen ○ Obligate anaerobes - oxygen is toxic to them ○ Facultative anaerobe - can grow with/without oxygen ○ Microaerophile - require oxygen to grow but at low levels ○ Aerotolerant - don’t use oxygen, but can tolerate it Understand how growth rate/generation time are determined. ○ What is the definition of growth rate and generation time? Growth rate - doubling of population per unit of time What equation? ○ k = n/t n = num of cells at end - start over t = time ○ k = ((log(b) - log(a)) /.3) / t Generation time - time required for all cells in a population to decide What equation? ○ g = t/n How do you determine generation time on a graph? ○ Look at the linear part ○ What is the relationship between growth rate and generation time? They are the reciprocal of one another Be able to identify the growth phases in batch culture and their characteristics ○ What are the 6 growth phases of a batch culture? Lag No cell division ○ You need replication of chromosomes for division Active metabolism ○ Turns on pathways  Adjustment period ○ If the bacteria sit for a period of time, what does this mean for the lag? It will be longer/slower Acceleration Increased growth rate Decreased generation time Exponential (log) Fastest growth rate, constant Linear on a log scale The growth rate is established by energy requirements for monomer synthesis The minimum doubling time for a medium is genetically defined ○ It depends on? How long it takes to make chromosomes Deceleration Decreased growth rate Increased generation time ○ What is happening to the cells? They are getting tried, nutrients has run out Cells run out of oxygen which leads to? Increase in toxins, pH goes down (more acidic) Stationary Heavily decreased growth rate Cells stop dividing ○ Do all bacteria die? No some bacteria grow and some die Metabolically active Death (not necessarily) Lysis Rarely linear Plateau in some media due to? ○ GASP - growth advantage in stationary phase phenotype If you added in a new medium, what could happen to the cells? They could restart the lag phase ○ Which stage is when cells maintain a steady state? During the linear part of the curve (exponential log phase) ○ Determine the phases of the batch culture on the graph  ○ What is the difference between rich and minimal medium? Rich medium - already have nutrients such as polypeptides, proteins, tryptine, and yeast extract, can begin the process sooner Minimal medium - need nitrogen sources such as NH4, proteins, and minerals, have to turn the pathways on to make them, which takes time Understand how viable cell counts are determined with their advantages and disadvantages ○ What are the 7 different ways to number viable cells? Microscopic counts What are the advantages and disadvantages to this? ○ Advantages - quick ○ Disadvantages - hard to count because you don’t know whether the cells are alive What is the solution to knowing whether the cells are alive or dead? ○ Fluorescent viability stain  What do the colors mean? Red - dead Green - alive ○ What are its disadvantages? It is difficult and time heavy Viable cell counts Serial dilutions ○ Bacteria culture is diluted in a series of steps to reduce the concentration of cells to a countable level ○ Plating ○ 1 mL from each serial dilution tube is placed on agar plate ○ Be able to distribute bacteria in individual colonies to count ○ ○ What are the advantages and disadvantages? Advantages - easier to count (green plate), simple Disadvantages - swarming makes it harder to count (red plate), time consuming, human error Turbidity  It measures how cloudy a bacterial suspension becomes as bacteria multiply, measured through absorbance (optical density) ○ Light passes through blank sample for the control ○ Bacterial suspension scatters the light, preventing it from reaching the detector. The more the bacteria, the more light is scattered Higher OD = more bacteria What are the advantages and disadvantages? ○ Advantages - quick, doesn’t disrupt the bacteria ○ Disadvantages - can’t tell if bacteria are alive or dead Which side has low turbidity vs. high turbidity? ○ Left = low Right = high Dry or wet weight Dry weight - measures mass of bacterial cells after water has been removed ○ Advantages and disadvantages Advantages - more accurate Disadvantages - time consuming (takes long to dry), can’t tell if bacteria are alive or dead Wet weight - measures mass of bacteria with water ○ Advantages and disadvantages Advantages - simple, fast, total biomass Disadvantages - inconsistent measurement of water in cells, can’t tell if alive or dead cells Colony size When bacteria spread on plate, cells multiply into colonies of different sizes/characteristics ○ You can look at 4 main properties of the cell Whole colony Edge Surface Elevation ○ Advantages and disadvantages Advantages - simple, easy to identify Disadvantages - subjective (people interpret a colony differently for classification), no dead/or alive cells, time consuming  Cell length Measuring bacteria based on length ○ Advantages and disadvantages Advantages- length indicates which stage of growth the cells are in Disadvantages - microscopy is a slow process Measurement of a product or activity You can look at ○ Total N or protein ○ O2 uptake ○ CO2 production ○ ATP production Be able to discuss the difference between batch and continuous culture and discuss the differences and similarities of chemostats vs turbidostats and when each might be used. ○ What is the difference between a batch culture and a continuous culture? Batch culture Closed culture system Limited nutrients Environment constantly changes ○ You would do this type of culture in what? A flask Continuous culture Open culture system Has a constant growth rate or cell density over extended periods ○ There is continuous maintenance for what type of conditions? Steady state ○ Define turbidostat - controlled by cell density Extra nutrients supplied What determines the rate of nutrients applied? Cell concentration Flow rate is adjusted to maintain specific turbidity levels When is it useful?  For obtaining a constant supply of identical cells ○ Define chemostat - controlled by nutrient concentration Medium is fed into culture at same rate it is removed One nutrient is limited, controlling biomass yield Rate of nutrient addition determines growth rate What does this allow for? ○ High density growth at low growth rates Example: clostridium thermocellum used to determine ethanol enhancement ○ What are the similarities and differences between turbidostat and chemostat? Similarities Continuous culture systems Regulate nutrient flow Differences Tubiodtate ○ Culture density controls nutrients ○ Limiting nutrient controls growth rate/yield Chemostat ○ Nutrients in excess ○ One essential nutrient limited Understand the difference between asynchronous and synchronous growth and methods to achieve synchrony in bacterial cultures. ○ What are the 2 types of synchronous growth methods? Induction What are the 2 types? ○ Temperature cycling - alternates temps at intervals ○ Phototroph light/dark cycling - alternates light and dark periods What does it do with nutrients and what type of inhibitors? ○ Withholds nutrients ○ Metabolic inhibitors Selection What are the 2 types? ○ Size - sucrose solution to create density gradient Cells will separate based on buoyancy and size Smaller cells will remain high Larger cells will sink ○ Age What are the 3 types? Funnel/Mesh - puts cells through membrane and filter with media Baby machine - newly divided cells come off ○ What happens with asynchronous population growth? Cells don’t grow logarithmically, ONLY exponentially Lecture 3 Amino acids and Proteins Understand the different types of proteins and how protein shape (conformation) is determined by their amino acid sequence. ○ What is the difference between simple and conjugated proteins? Simple - made of amino acids Conjugated - made with lots of things like carbs, lipids, nucleic acids, etc. ○ What proteins go with each type of macromolecule? Nucleic acids - nucleoproteins Lipids - lipoproteins Carbs - glycoproteins Prosthetic groups - heme, metalloproteins ○ What does conformation refer to? A protein's 3D shape What occurs when a protein is denatured? ○ Loss of bio activity ○ What are the 2 different types of protein conformation? Explain properties Fibrous H2O insoluble (hydrophobic) ○ Ex: collagen, keratin Structural Globular H2O double (hydrophilic) - water is on the outside, but not the inside Mobile /dynamic ○ The conformation is determined at 4 levels. Explain them  1 - covalent backbone and sequence of amino acids 2 - arrangement of polypeptide chain in space via one dimension a helix or B sheet ○ a helix has the same poly pep chain ○ B sheet has another poly pep chain 3 - bending or folding of chain for 3D Globular or fibrous structures ○ What stabilizes these structures? R groups Ionic bonding H bonds Disulfide bridge Hydrophobic interactions 4 - arrangement of individual polypep chains with two or more chains Oligomeric or multimeric ○ Oligomeric - multiple subunits Examples: RNA poly, ATCase, glutamine synthetase Understand the differences in amino acid classes and their general properties. ○ What are the parts that make up an amino acid? Chiral carbon Amino group Side chain Carboxyl group ○ What are the differences between positive and negative charges on the amino acid? Both positive - protonated 0 charge - Zwitterionic form (COO-) Both negative - deprotonated ○ What are the 4 classes of amino acids? Hydrophobic (nonpolar) Nonpolar side chains (aliphatic, aromatic) Avoid water Neutral (polar) Polar uncharged side chains Can form hydrogen bonds Positive (basic) at pH 7 Positively charged side chains Usually on exterior of proteins (react with water or negatively charged molecules like DNA/proteins) Negative (acidic) at pH 7 Negatively charged side chains Usually on exterior of proteins (react with water or positively charged molecules)  Be able to discuss the factors involved in protein function and how conformation is affected by physical parameters ○ What are the 8 main functions of proteins? Storage Transport Protective/structure Binding/regulatory Hormones/signaling Contractile Catalytic ○ What 4 physical parameters affect conformation of proteins? Temp High temps = denaturing Low temps = slow protein folding pH Ionic strength Stress Lecture 4 Enzymes; kinetics and allosterism Understand how the properties of the active site provide specificity and catalytic power. ○ What do enzymes have? High specificity and catalytic power ○ Explain difference between specificity and catalytic power Specificity - an enzyme catalyzes a single reaction or a set of related reactions Catalytic power - accelerates reactions by factors of as much as a million times Does this alter reaction equilibrium? ○ NO ○ What do substrates bind to? Active site What is the active site composed of? ○ Small portion of the enzyme ○ 3D entity - cleft or crevice How is specificity related? ○ The specificity of binding depends on the precise defined arrangement of atoms on the active site Are the substrates bound strong or weakly to the active site? ○ Weakly (by hydrogen bonds, ionic bound, hydrophobic interaction, van der waals, etc.)  What does the active site do to the substrate? ○ Places substrates in defined position (lock and key) Understand what KM is, its physiological significance, and why it is useful to know an enzyme's KM and Vmax. ○ What is Km? Constant that depends on S, T, pH, and ionic strength Physically means the Vmax/2 ○ What is the significance of Km? 4 reasons It establishes value for intracellular level of substrates The constant Km value provides a means for comparing the same enzymes from different organisms/types Km indicates suitability of alternative substrates for an enzyme A change in the value of Km can regulate enzyme activity ○ The lower the Km… The higher the affinity ○ What is Vmax? Max rate when all E (enzyme) sites are saturated with S (substrate) ○ What is kcat referred to as? Turnover number What is it also called? ○ k2, the catalytic constant ○ What is kcat/Km referred to as? Specificity constant, measure of how an enzyme converts substrate to product The higher the specificity constant… ○ The higher the specificity ○ Why is it useful to know an enzyme’s Km and Vmax? To help understand enzyme efficiency Compare enzyme performances, optimal conditions Drug design and inhibition Identify strategies used to control enzyme activity (inhibition types and features). ○ What are the 2 types of inhibition of enzyme activity? Irreversible Reversible What are the 3 types of reversible reactions? ○ Competitive  Substrate and inhibitor compete for active site Inhibitor binds to active site, preventing substrate from binding Reversible Vmax same, Km increases Ex: two cars competing for the same parking spot ○ Uncompetitive Inhibitor binds to ES complex Prevents enzyme from converting substrate into product Reversible Vmax decreases, Km decreases Ex: You get into the car, but someone puts spikes under the wheels ○ Noncompeitive Inhibitor binds to allosteric site (a site other than the active site) Changes enzyme shape/functionality Reversible/irreversible Vmax decreases, Km same Ex: steering wheel is locked. You can get in the car (bind to active site) but can’t drive the car ○ Understand what an allosteric enzyme is and how its sigmoidal kinetics is different from an enzyme that exhibits Michaelis-Menten kinetics. ○ What is an allosteric enzyme? Enzymes controlled at allosteric sites (sites other than the active site) who kinetics can’t be accounted for by the MM model What kind of kinetics is an allosteric enzyme? ○ Sigmoidal (s shaped) What causes the s shape to bend? ○ The allosteric enzyme’s activity controlled in small range around the Km What binding causes sigmoidal kinetics?  ○ Cooperative binding - binding of one substrate molecule increases affinity of the enzyme for another substrate ○ What is Michaelis-Menten kinetics used for? Understanding factors that influence rate of enzyme reactions E+S ES EP E+P ○ ES = enzyme substrate complex ○ EP = enzyme product complex What is k1 vs. k-1 involved with these? ○ k1 = forward reaction rate ○ k-1 = reverse reaction rate What happens to the rate of these reactions? ○ The rate increases with the substrate concentration ○ Understand the physiological importance of an allosteric enzyme and where they are generally found in pathways. ○ Allosteric enzymes can act as what 2 things? Activators or inhibitors ○ What does the allosteric activator stabilize? R state What does this do to the Km? ○ With activator, the Km is greater than Km without activator With increased affinity, the E is more responsive to lower S ○ What does the allosteric inhibitor stabilize? T state What does this do to the Km? ○ With inhibitor, the Km is greater than the Km without activator With decreased affinity, the E is less responsive to lower S ○ At what point in the pathways do these enzymes play a role in? Beginning of the path or branch routes Lecture 5 DNA: Structure and Function Understand the factors involved in initiation of replication of the bacterial chromosome. ○ Replication is the most important event in the cell cycle This process proceeds with what speed and accuracy? High speed and high accuracy ○ What does the oriC contain? AT rich DNA unwinding element (DUE) (the DNA complex unwinds at DUE creating single stranded region known as the open complex What is the difference between being AT rich and GC rich? ○ AT rich means 2 hydrogen bonds ○ GC rich means 3 hydrogen bonds = stronger/more stable DnaA binding sites ○ What else is at the oriC? 2 replication forks These forks have what type of replication? ○ Bidirectional (one strand is 5’ to 3’ and the other strand is 3’ to 5’) ○ What assembly is involved in initiation of replication? Two-state DnaA assembly model The assembly is in the presence of what? ○ ATP ○ What does the oriC do? (related to formation) oriC is an open complex formation ○ How is initiation of replication regulated? By 2 proteins DnaA = + factor (on switch) SeqA = - factor (off switch) Understand the replication process and the roles of different enzymes in synthesis and proofreading. ○ What are the 7 steps of replication initiation? Strand separation (pre-priming) Define the 3 important structures with this step ○ DnaC - Loading factor Brings DnaB to the region Once the helicase is on, what happens? ○ The DnaC falls of ○ DnaB - Helicase Unwinds helixes ○ SSB - single stranded binding proteins Maintains single stranded molecule, prevents reannealing and inhibits Dnases Unwinding  Define the 3 important structures with this step ○ DnaB - Helicase Unwinds DNA on the lagging strand ATP dependent Recruits DnaG to the fork ○ DnaG - Primase Creates short RNA primers to start DNA synthesis ○ DnaB + DnaG = primosome (ready to go!) Creates RNA primers on single stranded DNA Priming What is the main structure of this step? ○ DnaG - Primase Synthesizes complementary RNA primer necessary to prime DNA synthesis What is the primer structure? ○ 10 nucleotides long, become beginning of Okazaki fragments Elongation What is the main structure of this step? ○ DNA poly 3 Moves towards the replication fork in the 5’ to 3’ direction and adds complementary bases to 3’ hydroxyl groups a subunit = polymerase activity B subunit = forms sliding camp with a subunit which moves on template without being dislodged easily (donut shape wraps around DNA) Does 5’ to 3’ have errors? ○ Yes What is used to erase the mistakes? DnaQ subunit is a 3’ to 5’ exonuclease activity that erases mistakes ○ What are its properties? Proofreading function, 1 mistake/10^5 nucleotides What is the replisome? ○ Made of the primosome and DNA poly 3 Known also as the holoenzyme RNA primer removal and gap filling This primer removal and gap filling is carried out by what main structure of this step? ○ DNA poly 1  Uses RNase H activity to cleave RNA primer and 5’ to 3’ activity to fill in deoxyribonucleotides Ligation Formation of phosphodiester bonds between the 3' hydroxyl (–OH) group of one nucleotide and the 5' phosphate group of another nucleotide, resulting in a continuous strand of nucleic acid Termination Occurs at ter sites, 180 degrees opposite of the oriC at one of several locations ○ These ter sites contain what type of sites? What are they used for? Contain KOPS sites to orient DNA translocation by FtsK KOPS = FtsK orienting polar sequences Requires Tus proteins to bind ter sites asymmetrically and inhibit helicase What do topoisomerases and recombinases do in terminus regions? ○ Bind sites called Dif sequences for untangling DNA termini ○ What is introduced when you are unwinding and replicating DNA? Positive supercoils ahead of the fork What happens if there is excessive supercoiling? ○ It will slow fork movement What can remove positive supercoils? How? ○ DNA gyrase (toposiomerase 2+4) remove them by introducing negative supercoils ○ Summary of the 7 steps Strand separation What Happens: The double-stranded DNA unwinds to separate the two strands. This is the starting point for replication. Important Structures: ○ DnaC: Acts as a loading factor that helps bring DnaB (the helicase) to the DNA region that needs to be unwound. ○ DnaB (Helicase): This enzyme unwinds the DNA double helix, separating the two strands so they can be copied. ○ SSB (Single-Stranded Binding Proteins): These proteins attach to the single-stranded DNA and prevent the strands from re-annealing (coming back together) and protect them from degradation by nucleases. Unwinding What Happens: The helicase (DnaB) continues to unwind the DNA, preparing it for replication. Important Structures:  ○ DnaB (Helicase): Unwinds the DNA, specifically on the lagging strand, using energy from ATP. DnaG (Primase): This enzyme is recruited to the fork by DnaB to synthesize short RNA primers needed to start DNA synthesis. Primosome: This complex consists of DnaB and DnaG, and it’s now ready to start the replication process Priming What Happens: Short RNA primers are synthesized to provide a starting point for DNA synthesis. Main Structure: DnaG (Primase): Synthesizes the RNA primers that are necessary for DNA polymerase to start adding DNA nucleotides. Primer Structure: The primers are typically about 10 nucleotides long and serve as the beginning of the Okazaki fragments (short DNA pieces made on the lagging strand). Elongation What Happens: DNA polymerase III (DNA poly III) begins adding DNA nucleotides to the RNA primers, extending the new DNA strand. Main Structure: DNA Poly III: This enzyme moves toward the replication fork, adding complementary bases in the 5’ to 3’ direction (from the 3' end of the primer). Subunits: ○ A subunit: Responsible for the polymerase activity (adding nucleotides). ○ B subunit: Forms a sliding clamp (like a donut) that keeps the enzyme attached to the DNA template without falling off easily. Error Rate: Yes, mistakes can occur during DNA synthesis. Correction: DnaQ Subunit: Has a 3’ to 5’ exonuclease activity that removes mistakes, with a proofreading function that corrects errors at a rate of about 1 mistake per 100,000 nucleotides. Replisome What Happens: The replisome is the entire complex of proteins that is involved in DNA replication. Structure: It includes both the primosome (DnaB and DnaG) and DNA poly III, and is often referred to as the holoenzyme. RNA primer removal and gap filling  What Happens: The RNA primers are removed, and the gaps left behind are filled with DNA. Main Structure: DNA Poly I: This enzyme removes the RNA primers (using RNase H activity) and fills in the gaps with deoxyribonucleotides (DNA) Ligation: The final step involves forming phosphodiester bonds between nucleotides to create a continuous DNA strand. Know how Dam is involved in modifying DNA and its role in marking parent and daughter DNA strands. ○ As replication occurs, new strands are synthesized. How will new strands differ from the old strands? By degree of methylation Old templates of DNA are ____ vs. new templates of DNA are ___ ○ Methylated vs. hemimethylated (not methylated) ○ What enzyme methylates DNA? Dam enzyme (DNA adenine methylase) Understand the termination process and how intertwined chromosomes are resolved before being partitioned to daughter cells. ○ Termination Occurs at ter sites, 180 degrees opposite of the oriC at one of several locations Requires Tus proteins to bind ter sites asymmetrically and inhibit helicase Understand the bacterial cell cycle and its periods, being able to discuss how a bacterium can seemingly divide faster than it can replicate its chromosome. ○ What are the 3 periods of the bacterial cell cycle? B period Preparation time between cell division and beginning of DNA synthesis (division, replication initiation) Depends on how fast cell is growing ○ The B period is shorter if…. The growth period is fast C period Time from initiation through the end of replication (Z ring formation, end of replication) ○ It includes complete chromosomal replication About 40 min in e.coli D period Time between termination and cell division (septation, division) About 20 min in e.coli Replication initiation Know about the relationship of SeqA to DnaA and cell growth and their involvement in replication initiation.  ○ What does SeqA have a high affinity for? Hemimethylated DNA It prevents what? ○ From DNA binding there When does the binding occur? ○ After replication initiation delaying…. DnaA-ATP binding ○ What does the oriC region contain? 14 Dam sequences that overlap DnaA binding sites ○ What detaches from the oriC? Where? SeqA-oriC complexes detach during migration of oriC towards cell poles ○ Once replication is complete, what happens to the concentration of DnaA-ATP? It increases because DNA Poly 3 is decreased at oriC Lecture 6 RNA: Transcription Be able to describe the functional differences between RNA polymerase holoenzyme and core. ○ What are the functions of RNA polymerase holoenzyme vs. core enzymes? Holoenzyme - recognizes and binds to promoter regions What structures make it up? ○ Core enzyme and sigma factor Core enzyme - synthesizes RNA, but lacks ability to recognize specific promoter sequences What structures make up uo? ○ B, B’, a, and w subunits ○ How do the holoenzyme and core enzyme work together for transcription? Holoenzyme invites transcription by binding to specific sequences at the promoter while the core enzyme continuous elongation once the sigma factor drops off Understand promoter structure and the difference between strong and weak promoters. ○ What is the difference between strong and weak promoters? Strong promoters - closely match the consensus sequences at the -35 and -10 regions (more transcription) What does this match mean? ○ High affinity for RNa poly What types of genes are they found in? Give 2 examples of strong promoters ○ Found in genes that need to be transcribed at high levels ○ Examples: rRNA and tRNA  Weak promoters - do not match the consensus sequence (less transcription) What types of genes are they found in? ○ Genes that need tighter regulation or low levels of expression Know the role of the sigma factor and the involvement of alternative sigma factors in controlling gene expression. ○ What is the sigma factor? Subunit of the RNA poly holoenzyme ○ What is the function of the sigma factor? Directs enzyme to promoters by recognizing the -35 and -10 regions of the promoter, allows for binding of RNA poly ○ What do alternative sigma factors allow in controlling gene expression? Allow cell to respond to different environmental conditions What is an example of when sigma factors are activated? ○ During heat shock Understand the transcription process and mechanisms used to influence rate (class I vs. class II pause sites; NusA and NusB), editing/proofreading (GreA and GreB) and transcript length (termination). ○ What are the 3 steps of transcription? Initiation Elongation Termination ○ What 3 mechanisms influence the transcription process? Transcription/elongation rate What are the 2 components? ○ Class 1 and 2 pause sites What are their functions? Regulate speed of transcription What is the difference between class 1 and 2 sites? Class 1 sites - GC rich dyad (hairpin) Class 2 sites - GC rich sequence The class sites are influenced by what 2 elongation factors? ○ NusA - reduces pausing at class 1 sites ○ NusG - reduces pausing at class 2 sites Proofreading At class 2 pause sites, what can occur? ○ Proofreading or backtracking which in turn leaves what? Exposed mRNA ends What helps this? ○ GreA and GreB trigger RNAP endonuclease activity to remove the 3’ end of the transcript Transcript length (termination)  What are the 3 steps of termination? ○ Cessation of transcription ○ Release of transcript from ternary complex ○ Dissociation What are the two types? ○ Rho factor dependent Often at class 2 sites far from promoter at rho element (rut) Assisted by NusG ○ Rho factor independent Often at class 1 sites where hairpin destabilizes the structure (can be 10-90% effective) Assisted by NusA Be able to discuss how transcription initiation is controlled and understand differences between negative and positive control (class I and class II activators). ○ What is the difference between negative and positive control? Negative control - repressors interfere with transcription initiation by binding at or near the promoter What does it alter? ○ RNAP so it can’t recognize promoter classes ○ Replaces sigma with a different sigma Positive control - activators that direct interact with RNAP to influence transcription initiation Where do class 1, class 2, and conformation activators bind? ○ Class 1 - upstream of promoter ○ Class 2 - site adjacent to or overlapping -35 ○ Conformation - between -10 and -35 Be able to distinguish RNA classes (stable vs. "unstable”) and their differences. ○ What are the 2 RNA classes? mRNA - products that are directly used for protein synthesis Unstable Associated with weak promoters tRNA/rRNA - RNA products that are not translated into proteins Stable Associated with strong promoters ○ What is proportional to growth rate for stable RNA? [rRNA] and [tRNA] are proportional to growth rate Be familiar with factors involved in the stringent response, how they are made and how they act (relA vs. spoT) compared to the relaxed response and the role of DksA and discriminator sequences in transcription initiation. ○ What occurs during stringent response? Collective and highly specific drop in expression of stable RNA genes What is an example? ○ Global control mechanism  ○ When is stringent response triggered? Nutrient starvation What 2 proteins regulate this response? ○ RelA and SpoT What is the difference between RelA and SpoT? RelA - activated on the ribosomes when uncharged tRNA is detected ○ It leads to the production of? ppGpp (signaling molecule) SpoT - can synthesize or degrade ppGpp ○ What does this allow for? Fine control of the response ○ What are the 4 stringent responses to starvation conditions? 10 to 20 x decrease in stable [RNA] 2 to 2 x decrease in mRNA production Increase in biosynthetic pathway mRNA Decrease in protein synthesis ○ What do DksA and discrimination sequences do? Modulate RNA poly activity in response to cell changes ○ What is a relaxed response? When there is a lack of regulation under nutrient rich conditions Be familiar with RNA processing—the role of RNases, ribozyme and base modifications. ○ What role do RNases, ribozymes, and base modifications play in RNA processing? RNases - enzymes that degrade RNA, regulating RNA stability What do different RNases do? ○ Act at specific sites or degrade DNA in a sequence specific manner Ribozymes - RNA molecules with catalytic activity What type of processes would they be involved in? (2) ○ Splicing and cleavage of RNA precursors Base modifications - occur on stable RNAs (tRNA, rRNA) They are essential for what 2 things? ○ Proper folding and function of RNAs What is an example of base modifications? ○ Methylation (add methyl groups to molecules) Lecture 7 Translation Understand how tRNAs are activated (charged) with amino acids and the involvement of “common” tRNA synthetases as well as the charging of "unusual" tRNAs. ○ How are tRNAs activated (charged) with amino acids?  tRNAs activated or charged by aminoacyl-tRNA synthetases What do these enzymes do? ○ Attach the correct amino acid to its corresponding tRNA Explain the 2 step process 1. Amino acid activated by ATP, forming aminoacyl-AMP 2. Amino acid transferred to the 3’ end of the tRNA What is a property of the tRNA synthetase? Highly specific for both the amino acid and the corresponding tRNA Be familiar with the proofreading and editing processes in tRNA activation and mRNA translation. ○ Describe the proofreading editing process in tRNA activation During tRNA charging, aminoacyl tRNA synthetases have proofreading abilities to make sure the correct amino acid is attached to the tRNA What happens if an incorrect amino acid is attached? ○ The enzyme’s editing site removes the mischarged amino acid ○ Describe the proofreading editing process in mRNA translation The ribosome checks for codon-anticodon pairing between the mRNA and tRNA to ensure accuracy What happens if there is a mismatch? ○ Elongation is paused and the incorrect tRNA is rejected, preventing errors in growing chain Like a quality control inspector that removes defects from production Understand ribosome assembly as it relates to the translation process (i.e., the role of 50S and 30S subunits and the function and location of 23S, 5S, and 16S rRNAs). ○ What is the role of the 50S and 30S ribosomal subunits and their associated rRNAs in the translation process? 50S - contains 2 RNAs (23S rRNA and 5S rRNA) Describe the functions of these 2 RNAs ○ 23S rRNA - catalyzes peptide bond formation ○ 5S rRNA - stabilizes ribosome structure 30S - contains 1 RNA (16S rRNA) Describe the function of this RNA ○ Helps the ribosome recognize the Shine-Dalgarno sequence on the mRNA ○ What do the 50S and 30S subunits form? What does this do? 70S ribosome, it moves along the mRNA to synthesize proteins Be familiar with the translation process and roles of initiation, elongation and release factors.  ○ What are the roles of initiation, elongation, and release factors in translation? Initiation factors What are the 3 initiation factors? ○ IF-1, IF-2, and IF-3 What do these initiation factors do? ○ Assembles the ribosome at the start codon What does IF-2 specifically do? Binds GTP and helps position the initiator tRNA at the start codon (fMet-tRNA) Elongation factors What are the 2 elongation factors? ○ EF-Tu and EF-G What does each elongation factor do? ○ EF-Tu - brings aminoacyl-tRNAs to the A site of ribosome ○ EF-G - moves ribosome along the mRNA through GTP hydrolysis Release factors What are the 2 release factors? ○ RF1 and RF2 Describe situation when these factors are used ○ When a stop codon is encountered, release factors bind to the ribosome and cause the release of the new protein ○ from the ribosome. The ribosome dissociates to end translation Understand the involvement of GTP and GDP in the translation process and their relationship to the stringent response (rprotein L11 and RelA). ○ What is the role of GTP in translation? GTP provides energy for what 3 processes? tRNA position Translocation Termination What happens to GTP during these processes? GTP is hydrolyzed to GDP ○ Why is this needed? To change the conformation for ribosome proteins and EF ○ Describe function of L11 and RelA in the stringent response The ribosomal protein L11 interacts with RelA, which detects amino acid starvation What does RelA synthesize? ○ (p)ppGpp which downregulates rRNA synthesis This shifts the cell's priorities from ____ to ____ during ____?  Growth to survival during stressful conditions Be familiar with the consequences of different mutations on gene products. ○ What are the potential consequences of different mutations on gene product? Silent mutations Don’t change the amino acid sequence due to redundancy of genetic code (multiple codons for one amino acid) Missense mutations Substitution of one amino acid, may affect protein function Nonsense mutations Introduces premature stop codon, leading to a truncated (shortened) protein Frameshift mutations Caused by insertions or deletions that shift the reading frame, usually resulting in a different protein Be familiar with how selenocysteine is incorporated into proteins, the SECIS signal and its relationship to stop codon. ○ Where is selenocysteine incorporated into proteins? UGA codons (stop signals) ○ What is the role of the SECIS signal? SENIS (selenocysteine insertion sequence) allows the ribosome to interpret UGA as a signal to insert selenocysteine instead of terminating translation Where does tRNAsec come in? ○ The tRNAsec is charged with selenocysteine and guided to the ribosomes by EFs Understand the evidence for autogenous regulation (and against gene dosage) of ribosome synthesis (Nomura’s experiments). ○ What evidence supports autogenous regulation of ribosome synthesis, according to Nomura’s experiments? Nomura’s experiments showed that ribosomal protein synthesis is regulated through autogenous feedback What is autogenous feedback? ○ Certain ribosomal proteins bind to their own mRNA to inhibit translation When does this occur? When there is an excess of ribosomal proteins relative to rRNA ○ The findings provided evidence against what model? Explain Gene dosage model, which suggested that ribosome synthesis is controlled by the number of gene copies BUT we know that ribosomal proteins instead regulate their own production Lecture 8 Regulation of gene expression; transcriptional control Understand the difference between constitutive and inducible pathways and sequential vs. coordinate induction. ○ What is the difference between constitutive and inducible pathways? Constitutive pathways - are always “on” and active The proteins or enzymes are constantly being produced ○ Example: proteins produced for cell survival such as in glycolysis pathways Inducible pathways - only activated under certain conditions Example: lac operon in e.coli is only turned on when lactose is present and glucose is absent/run out ○ Lac operon allows the cell to use lactose as an energy source, but ONLY when necessary ○ What is the difference between sequential and coordinate induction? Sequential induction - occurs when multiple genes are turned on in a specific sequence It depends on? ○ The availability of substrates or presence of intermediates Ex: enzymes in a pathway may be induced one after the other as their substrates become available Coordinate induction - occurs when multiple genes are simultaneously activated by the same signal or environmental change Ex: lac operon, several genes simultaneously activated in response to lactose Know the similarities and differences between positive and negative control mechanisms. ○ Already talked about differences above ○ What are the similarities? Both control mechanisms regulate gene expression by affecting whether RNA poly can access the promoter and initiate transcription The difference lies in whether the control mechanism inhibits or enhances transcription Understand specific (LacR, AraC) and general (CAP) controls for lac and ara and be able to explain the phenotypes for mutants and lactose/glucose growth physiology. ○ How do LacR and AraC regulate the lac and ara operons? LacR (Lac repressor) - in the absence of lactose, LacR binds to the operator of the lac operon, preventing transcription. When lactose is present, it binds to LacR, causing it to release from the operator and allowing transcription to proceed What would the phenotypes for mutants be of LacR?  ○ A mutant with a defective LacR that cannot bind lactose would always repress the lac operon, even when lactose is present, leading to a phenotype that cannot use lactose AraC (Ara Repressor/Activator) - regulates the arabinose operon in a dual manner. When arabinose is absent, AraC acts as a repressor, preventing transcription. When arabinose is present, AraC acts as an activator, promoting transcription What would the phenotypes for mutants be of AraC? ○ A mutant with a defective AraC wouldn’t be able to induce the arabinose operon, resulting in a phenotype that can’t use arabinose ○ How does CAP influence these 2 systems? CAP is a global regulator that works with cAMP to promote operon transcriptions (like the lac operon when glucose levels are low) What does CAP bind to? ○ The promoter region of the operon, enhancing RNA poly binding and transcription when glucose is absent What would CAP mutants look like? ○ Even when glucose is absent and lactose is present, the lac operon would be be efficiently transcribed as CAP cannot activate the operon without functional CAP Know the factors involved in catabolite repression and their importance/role. ○ Catabolite repression involves the regulation of? Gene expression based on carbon source availability What is a preferred carbon source? ○ Glucose The presence of a preferred carbon source inhibits what? ○ The expression of operons responsible for metabolizing less preferred sources like lactose or arabinose What are the 3 key factors of catabolite repression? Glucose - when glucose is present, it is preferably used for energy and transcription of operons for other sugars is repressed cAMP and CAP - when glucose levels are low, the levels of cAMP increase. cAMP binds to CAP, which binds to specific promoter regions, enhancing transcription of operons involved in metabolizing other sugars Be able to explain the "glucose effect" (carbon catabolite repression) and how it can be observed in growth experiments. ○ What is the “glucose effect”? Carbon catabolite repression, where the presence of glucose represses the expression of genes involved in the metabolism of other sugars This effect can be observed in growth experiments such as? Explain ○ Diauxic growth  In a growth curve, cells grow rapidly on glucose (preferred carbon source) Once glucose is gone, there is a lag phase as the cells adjust their metabolism to begin using the non-preferred carbon source (lactose) After the lag phase, growth resumes as a slower rates as the non-preferred carbon source is used Lecture 9/10 Regulation of gene expression; post-transcriptional/translational control Understand how attenuation works for amino acid biosynthetic operons and be able to explain the difference between trp and his end-product repression mechanisms ○ How does attenuation work for amino acid biosynthetic operons? It controls gene expression by influencing formation of secondary structures in mRNA leader sequence ○ Explain trp operon In the trp operon, tryptophan acts as a… Corepressor - binding to TrpR repressor protein which… ○ Blocks transcription when tryptophan is abundant What does the trp operon use? Attenuation, where the formation of a 3:4 stem-loop structure in mRNA leader sequence terminates transcription early when tryptophan is in excess ○ Explain his operon What does this operon lack? Repressor proteins ○ So it relies entirely on what? Attenuation What occurs under histidine starvation? Ribosome stalling occurs at mRNA leader sequence, allowing a 2:3 stem-loop to form ○ What does this prevent the formation of? What does it enable? Prevents terminator structure (3:4 loop) Enables continued transcription Understand the general properties of feedback inhibition and allosteric control and how they relate to linear and branched pathways. ○ What are the general properties of feedback inhibition? Mechanism where the end product of a metabolic pathway inhibits activity of the first enzyme in the pathway  What does this prevent? ○ Overproduction of product ○ What are the general properties of allosteric control? Binding an effector molecule at a site other than the active site (allosteric site) What does this cause? ○ Conformational changes that either can… Enhance or inhibit enzyme activity ○ How does feedback inhibition relate to linear pathways? Feedback inhibition usually targets the first enzyme in the sequence What type of enzyme is the first in the linear pathway? ○ Allosteric ○ How does feedback inhibition relate to branched pathways? Feedback inhibition is more complex, each branch has its own control mechanisms What type of enzymes act at key points in branched pathways and what do they do? ○ Allosteric enzymes ○ Coordinate metabolic flux ○ All the enzymes in the pathways are under allosteric control Know how covalent modification can be used to control an enzyme’s activity and types of covalent modification. ○ How does covalent modification regulate enzyme activity? It regulates enzyme activity by adding or removing chemical groups When it adds or removes these groups, what can it alter? ○ Enzyme’s structure and function ○ Is covalent modification reversible or irreversible? Reversible ○ What are types of covalent modification? (reversible modification) Phosphorylation Adds phosphate groups by kinases to (de)activate enzymes Changes conformation ○ Reverse reaction: adds phosphatase to remove phosphate groups Adenylation Adds AMP to enzyme For tyrosine Methylation and acetylation Adds methyl or acetyl groups ○ What do these modifications allow the enzymes to do within the environment? Adapt quickly to changes  Know what specific activity is and how it can be used to determine an enzyme’s activity level under different physiological conditions and whether its control occurs at the transcriptional or post translational levels ○ What is specific activity? Ratio of enzyme activity to the amount of total protein in a sample What does it reflect in regards to the enzyme? ○ Reflects how much active enzyme is present relative to total ○ What does the measuring specific activity under different conditions of enzymes determine? Whether changes in enzyme levels are due to transcriptional or post translational control What is the difference between transcriptional and post translational control? ○ Transcriptional - change in the amount of enzyme produced ○ Post translational - change in enzyme activity without changes in protein levels If specific activity changes with a change in enzyme concentration, what type of regulation does it suggest? (translational or post translational) ○ Post translational Know the role of N-degron and C-degron proteolysis signals and how they are used in regulation and clearing damaged proteins. ○ What are N-degron and C-degron proteolysis signals? They are degradation signals located at the N or C terminus of proteins Where do these signals mark proteins for degradation? ○ Proteasome What is the proteasome responsible for? Breaking down damaged, misfolded, or excess proteins ○ What is the purpose of the degradation process with proteins? To ensure proper protein quality control and regulates their levels ○ If you tag proteins with N or C degron signals, what occurs (specifically vs. big picture? Cell can remove defective proteins Thereby maintaining homeostasis ○ What is the role of proteolysis in the cell? Breakdown of proteins, particularly during times of stress or starvation What are the 2 key functions of proteolysis? ○ Nutrient recycling During starvation, proteolysis increases, allowing the cell to recycle aa ○ Clearing damaged proteins  Proteolysis removes incomplete/abnormal proteins from damaged mRNA or mutations, preventing accumulation of non-functional proteins Acts like a garbage disposal ○ When does proteolysis play a role in maintaining homeostasis? During stress conditions like starvation or when damaged proteins accumulate How does proteolysis help? ○ Provide amino acids ○ Remove abnormal proteins ○ Regulate cell processes