Exam 3 Flashcards
Aerobic Respiration
reduces oxygen
Anaerobic respiration
reduces anything but oxygen
Geobacter Metallireducens
- Consumes sugars and oil-based pollutants
- Respires metals including Fe, U & Tc
- Bioremediation
- Respires insoluble molecules
- Pili conduct electrons outside of cell (nanowires)
- Biofuel Cells
- Nanowires: allows for respiration of insoluble molecules, conductive pilus anchored in the plasma membrane and part of the ETC
Lithotrophy
Eat inorganics Breathe anything (electrons)
Obtaining electrons by consuming inorganic molecules
Organotrophy
Eat organics Breathe anything (electrons)
Hydrogen Oxidation (How is ATP, NADH & PMF generated, how do electrons move through tower, example of bacteria/system)
ATP: F1F0 ATPase
NADH: Hydrogenase => direct reduction of H2 Gas
PMF: ETC, reduction of O2 => H2O
Tower: Move down H2 => O2
Ex: Subsurface Lithotrophic Microbial Ecosystem (SLiME), Chemolithoautotrophs, require nothing but gas to grow
Sulfur Oxidation (How is ATP, NADH & PMF generated, how do electrons move through tower, example of bacteria/system)
ATP: F1F0 ATPase NADH: Reverse ETC PMF: ETC Tower: Up S/H2S => NAD+/NADH Ex: Hydrothermal Vents
Iron Oxidation (How is ATP, NADH & PMF generated, how do electrons move through tower, example of bacteria/system)
ATP: F1F0 ATPase NADH: Reverse ETC PMF: ETC Tower: Up Fe3+/Fe2+ => NAD+/NADH Ex: Lepthothrix bacteria- grow in biofilm in saturated soils with high levels of iron
Adv/ Disadv of Iron Oxidation
Adv: Iron so low in energy that few other bacteria bother to compete, Iron is one of the most abundant resources on earth
Disadv: Takes A LOT of electrons for a little energy, growth could be fantastically slow, doubling times of months or more
3 kinds of phototrophy & what kinds of energy do they produce
Cyclic: PMF (ATP)
Acyclic: NADH, PMF (ATP)
Bacteriorhodipsin: PMF (ATP); does not use ETC
What is cyclic phototrophy electron donor?
Chlorophyll
What is cyclic phototrophy electron acceptor?
Chlorophyll
What is the role of chlorophyll
cofactor that absorbs light
What is the purpose of the reaction centers
holds an array of chlorophyll molecules
How does electron transfer in cyclic phototrophy work
excited electron passed to membrane carriers, ETC makes PMF, electrons can return to any reaction center
How does making NADH break the cycle
electrons must be filled from another source to complete the cycle
How does acyclic phototrophy “fix the cycle”
electrons filled from another source to complete the cycle
Two methods used by phototrophs to make NADH
Purple/green sulfur: bacteria w strong rxn centers w enough energy to directly reduce NAD+
Purple/green nonsulfur: bacteria w weak rxn centers and must use reverse ETC
How does the oxygenic acyclic phototrophy system use water to produce NADH
Links 2 different photosystem rxns together, coupled in series
First photosystem has enough resting potential to accept electron from H2O
Second photosystem has enough activated potential to directly reduce NAD+
“eats H2O, gives off O2”
What are the ways to increase the surface area of the rxn centers to more efficiently use light?
1) Membrane Invaginations: increase SA to mount reaction centers
2) Phycobiliproteins: antenna proteins that funnel energy to rxn centers
What is the solution to having too much light energy
more light energy to carotenoids (cofactors that absorb light energy)
What contributes to bacteria having different pigments
Phototrophic bacteria have a wide range of absorbance profiles
Different organisms have evolved different light harvesting pigments
Halobacterium salinarum
Archaeon, extremely halophilic, desiccation/ radiation resistant, very simple photosystem (bacteriorhodopsin), single proton pump directly coupled to light absorption and does not use ETC
How do bacteriorhodipsins work
Reaction center directly coupled with proton pump
From what doe Heterotrophs obtain carbon? What pathways are used?
Organic chemicals: C and N enter macromolecules as reduced carbon compounds like AA
Pathways: Glycolysis, PPP, TCA cycle
What macronutrients in the atmosphere can be fixed
Carbon (CO2)
Nitrogen (N2 gas)
H, O (H2O)
Definition of Autotrophy
Building reduced carbon (sugars) directly from CO2
How are NADH and NADPH used differently
Both have same function to donate electrons
NADH: Catabolism
NADPH: Anabolism
What enzyme enables carboxylation of 5 carbon ribulose
RuBiSCO: Ribulose Bisphosphate Carboxylase
3 Steps in carbon fixation
1) Carboxylation: Attaches one CO2 molecule to end of 5C sugar
2) Reduction: “fixes” carbon ation biologically
3) Rearrangement: 1 Carbon donated to make glucose each time.
How much energy is used in carbon fixation
18 ATP, 12 NADPH (6x through cycle)
What happens when CO2 is low? Whats the solution?
O2 can compete for the enzyme and be incorporated producing toxic glycolic acid (“phosphorespiration”)
Solution: Carboxysomes (protein shells that contain a lot of RuBiSCO, enrich for CO2 and exclude O2)
Compare and contrast Chemoorganoheterotrophy and Photoautotrophy
Chemoorganoheterotrophy: Glycolysis/TCA cycle, release PMF, release ATP/NADPH, aerobic respiration
Photoautotrophy: Oxygenic acyclic phototrophy, take in ATP/ NADPH, take in PMF, Autotrophy
How does nitrogen enter the cell
1) Directly through premade AA
2) As ammonia attached to Glutamate (AA)
What are the 2 enzymes involved in the 2 step glutamine synthesis pathway
Glutamine Synthetase and Glutamate synthetase
Wha tis the enzyme involved in the one step glutamate synthesis pathway
Glutamate Dehydrogenase
Where does reduced Nitrogen (ammonia) come from?
2) Geothermal
What is nitrogen fixation
Biological conversion of N2 => NH4+
Strictly a bacterial process but not all bacteria can do it
How much energy is used in nitrogen fixation
Extremely energetically expensive
4 NADPH
18 ATP
What proteins are involved with nitrogen fixation
Nitrogenase enzyme
Fe protein: delivers electrons
FeMo protein: reduces nitrogen
What is the problem that stems from atmospheric concentrations of N2 and O2
Nitrogenase destroyed by oxygen, Active site of iron protein exposed to cytoplasm, O2 reacts with iron, iron in protein rusts
What are the solutions that have evolved to solve O2 problems in nitrogen fixation
1) Anaerobic conditions: no O2 present
2) Extremely rapid respiration: respire O2 so fast that the concentration of O2 is low
3) Heterocysts: specialized, differentiated, non-growing, O2 impermeable cell types found in cyanobacteria.
Oxygenic phototrophy in most of the chain, heterocysts fixes nitrogen and shares with chain
Sinorhizobium meliloti
Legume symbiont
Fixes N2 in plant nodules
different legumes form symbioses with diff bacteria
Bacteriod does not grow
Plant gets N2, bacterium gets carbon (sugars)
How does cell-cell communication play a role in legume symbiosis
plant root sends flavanoid signal to attract bacteria, bacteria sends signal “Nod factor” back to plant.
Signals are specific for both plant and bacterium, ensures proper recognition between partners
What does the plant provide in legume symbiosis
Carbon (sugars)
What does the bacteria provide in legume symbiosis
Nitrogen (NH3)
How does this legume symbiosis benefit the group if the bacterioid cannot grow?
Rhizopines (check?)
How are lipid subunits activated
Attaching Acyl carrier protein (ACP)
What do the primer and extender do?
Primer: often Acetyl-ACP (2C) but can vary
Extender: always malonlyl-ACP (3C) => donates 2C to primer, elongates chain by 2C
How do different types of fatty acids get made? (even #, odd #, branched)
Different primers!
Acetyl-ACP: Chains w even #C
Propionyl-ACP: chains w odd # C
Isovaleryl-ACP: chains w odd # chains w branch
How does unsaturation of fatty acids occur
1) Skip reduction step: reducing enzyme fails at low temps so that double bond is preserved during elongation
1) Monooxygenase: enzyme made at lower temps and adds oxyygen to reintroduce double bond after chainis completed
What AA is attached to glycerol-phosphate backbone in the phospholipid phosphatidylserine (PS)
Serine
What is removed to make phosphatidylethanolamine (PE)
CO2
How are sugar monomers activated
attaching nucleotides (UDP)
How do polysaccharides polymerize and where?
Glucose adds to glycogen chain and UDP released into cytoplasm
How are peptidoglycan monomers activated
1) NAM activated w UDP
2) 5 AA attached sequentially to NAM (pentapeptide)
3) NAM-Peptide is transferred to membrane carrier lipid, lipid carrier becomes new monomer activator
4) NAG activated with UDP
5) NAG Attached to NAM-penta peptide
6) Lipid carrier and bound NAG/NAM-pentapeptide flipped to opposite side of membrane (catalyzed by flippase)
How is peptidoglycan monomer polymerized?
7) NAG/NAM polymerized onto existing chain in wall
How are the chains crosslinked in peptidoglycan
8) Pentapeptide crosslinked by penicillin binding protein (PBP)
What steps does penicillin inhibit
8) crosslinking
What step does Vancomycin inhibit?
7) Polymerization
What steps do Bacitracin inhibit
1, 4) UDP activation
How doe scientists observe peptidoglycan synthesis
Study fluorescently labeled vancomycin wherever they saw fluorescence, synthesis was happening
Where did scientists see peptidoglycan synthesis? What were the surprises?
Synthesis is localized, not random, 2 patterns of synthesis
What were the two patterns of peptidoglycan synthesis?
1) As cell elongates, new wall inserted as spiral (MreB)
2) as cell divides, new wall inserted as ring (FtsZ)
What is the central Dogma
DNA (Transcription =>) RNA (Translation =>) Protein (replication =>) DNA
What are the protein synthesis subunits? How many are there
20 Amino Acids
How are the amino acids activated? What enzyme is involved?
tRNAs -> each AA matches a tRNA w specific nucleotide sequence.
Enzyme= Amino Acyl-tRNA synthetases: recognizes proper AA and matches tRNA by anti codon and side loops
What enzyme synthesizes proteins?
Ribosome: 3 diff rRNAs, 55 diff proteins
What are the components of the ribosome
30S rRNA, 50s rRNA, and proteins
What role does mRNA play in translation
template for protein synthesis
How is mRNA made
transcribed by RNA polymerase from DNA Sequence
How is translation initiated? What sequence is required? What enzymes are required
1) Ribosome assembles on mRNA at specific ribosome binding site (SD sequence)
2) IF2 (Initiation Factor 2) delivers first activated tRNA to ribosomes
3) EF-tu (elongation factor Tu) delivers all other activated tRNAs to ribosome
What is the Shine-Dalgarno Sequence
Where the ribosome binds
How are peptide bonds formed
Catalyzed by 23s rRNA in ribosome
AA bond to tRNA by carboxyl groups, Amino (N) of AA attacks carboxyl (C) of another, proteins synthesized N -> C terminus
What direction is mRNA template read? What direction is protein synthesized?
mRNA-> 5’ -> 3’
Protein N-C
WHat enzymes are required for elongation
EF-G (Elongation factor G) slides ribosome 3 base pairs downstream on mRNA
What enzymes are required for termination
Release Factors (RF proteins): recognize stop codons and arrest protein synthesis
What does the antibiotic tetracycline do
blocks amino acyl tRNA entry
What does the antibiotic chloramphenicol do
blocks peptide bond formation
What does the antibiotic erythromycin do
blocks ribosome translocation
How is the protein sequence determined
Each protein templated by unique mRNA w particular sequence
What is the info contained in the mRNA sequence
1) Ribosome binding site
2) Translational start site
3) Code for protein translation
4) translational stop site
How is the start codon recognized
3’ end of 16s rRNA base pairs w complementary site (SD) 6-9 base pairs downstream of SD is translational start site. First AA is ALWAYS f-Met in bacteria, start codon is AUG (also UUG and GUG)
What is a codon
Series of 3 nucleotides that specify particular AA, initiation codon sets frame which determines all subsequent codons
Why use a 3 nucleotide instead of 2 or 1
enough possibilities for all AA to have a codon w some to spare
How does the cell deal with degeneracy
1) one tRNA recognizes multiple codons by loose specificity in 3rd base “wobble position”
2) Make a second tRNA for the same AA to recognize particular codon
What are stop codons
3 codons that do not code for AA, instruct ribosome to stop translation, recognized by release factors: terminate translation and dissemble ribosome
What are the 3 functions of SD sequence
1) Ribosome binding site
2) Determines start codon
3) Controls protein copy #: more similar SD is to a perfect match w 16S rRNA, more frequently ribosomes initiate and more protein is translated
What are polysomes and why can they from in bacteria
Multiple ribosomes on the same template, can occur bc transcription and translation are coupled in bacteria
Compare and contrast bacterial and eukaryotic translation
1) B: Ribosome recognizes SD sequence
E: Ribosome recognizes 5’ cap modification
2)B: Multiple initiating codons (AUG, UUG, GUG)
E: one initiating codon (AUG)
3) B: f-Met
E: Met
4) B: Transcription and translation occur simultaneously
E: Transcription and translation occur sequentially
5) Different ribosome structure
B: 70S ribosomes 16S rRNA
E: 80S ribosomes, 18S rRNA
Three examples of Chaperones
DnaK: help fold proteins as they emerge from the ribosome, activated at high temps, part of the Heat Shock Response, helps limit damage
SecB: prevent proteins from folding for delivery to secretion machinery
GroEL: give proteins a second chance to refold if misfolding has already occurred. Activated at high temps to refold denatured proteins
What are proteases? Example?
Destroy misfolded proteins and recycle AA
Ex: ClpCP
What are the three classes of RNA molecules and how do they relate to translation
How are RNA molecules polymerized in transcription
1) mRNA: informational RNA, has SD sequence, One specific mRNA for each protein to be translated
2) tRNA: activates AA, decodes mRNA, shared pool, no SD or start codon
3) rRNA: major structural and catalytic component of ribosomes. Shared pool, no SD or start codon
How are nucleotides activated in transcription
Phosphorylation
ADP -> ATP
How are RNA molecules polymerized in transcription
5’ -> 3’
What enzyme perfomes transcription? Components of the enzyme?
RNA polymerase
4 complex proteins make up core: a, a, B, B’ (responsible for catalyzing polymerization of RNA)
sigma: determines which promoters RNAP binds to and controls specificity
Core + Sigma = Holoenzyme
What are promoters
DNA sequences that indicate where to start transcription
What are sigma factors
proteins that bind specific promoter sequences and instruct RNAP where to begin transcribing. Bind at -35 and -10 box
What sequence is important for sigma factors
consensus sequence
What is a consensus sequence
average of all promoters recognized by a sigma factor
How are 2 strands of DNA separated
RNAP melts a bubble of dsDNA to single strands
What is the pairing of nucleotides bn RNA and DNA
A=U/T
G=C
Which strand is the template strand in DNA
bottom DNA strand
What does Rho-dependent termination require
Rho protein binds to untranslated mRNA, rolls along transcript and knocks RNAP off.
Rho binds to rut sites (C bases w regular periodicity)
ATP must be consumed, energy dependent process, ribosomes block rut sites normally
What does Rho-independent termination require
Terminated by sequence in RNA itself. “Hairpin” stable stem loop followed by poly U tract knocks RNAP off DNA template and halts transcription
What is a gene/cistron
Segment of DNA that codes for a functional product for each gene, multiple transcripts are made depending on promoter consensus
What is an operon
when multiple genes are expressed from common promoter, each gene has its own SD, translational start/stop
when multiple genes are expressed from common promoter, each gene has its own SD, translational start/stop
When translational start/stop sequences of 2 adjacent genes overlap. Instead of ribosome falling off transcript, it backs up and begins translating again. Ensures “translationally coupled proteins” are always made together and at the same copy number.
How are tRNAs and rRNAs encoded on the genome? What does RNase III do?
Coded in operons, encoded on same transcript.
RNase III enzymatically cuts to separate
What are alternative sigma factors
Different sigma factors recognize diff promoter sequences. Diff genes can be expressed by changing sigma factors
How do alternative sigma factors differ from sigma A?
Diff consensus sequences, recognize diff promoter sequences, express diff genes
Bacillus subtilis
Industrial workhorse: produces proteases, riboflavin, fungicide, powerful genetic system
“Component” takes up free DNA from the environment
sporulation, cascade of sigma factors: certain sigma factors activate others
Spore is metabolically dormant, resistant to heat, desiccation, and radiation
What are activators and how do they work
Proteins that bind to specific DNA sequences and activate expression from downstream promoters
Often compensate for a weak -35 sequence
Lac Operon: How does CAP function?
Activates lac operon transcription by binding to a specific DNA sequence but requires cAMP to do so.
Lac operon: what does cAMP do
Activates CAP and is synthsized by adenylate cyclase. Enables quaternary structure, acts as glue bn dimers
Under what conditions are the lac genes transcribed and how does CAP influence this
transcribed when lactose is present and glucose is not present. CAP inhibited by glucose which also inhibits cAMP
What are repressors and how do they work
proteins that bind to a specific DNA sequence and inhibit gene transcription. Repressor binding overlaps promoter and blocks RNAP access
How does LacI function
Inhibits transcription unless lactose is present. Severely bends DNA and prevents access of RNAP to lac promoter. LacI inhibited by lactose.
In two component systems, what is HPK and what does it do
Histidine Protein Kinase receives signal, then autophosphorylates and transfers phosphate to an aspartase on RR
What is the RR, what does it do?
Response Regulator that controls gene expression. Phosphorylation of RR activates DNA binding and RNAP recruitment
Fremyella diplosiphon
Cyanobacterium that fixes carbon and nitrogen. Heterocysts protect nitrogenase. Oxygenic phototrophy, makes gas vesicles, Chromatic acclimation: controlled by 2 component system
HPK: RcaE RR: RcaC
In green light: makes green absorbing pigment (default)
In red light: RcaC phosphorylated and binds to DNA activates red light and supresses green light
What is a riboswitch and what are two ways that it functions
RNA sequence pattern in 5’ leader of mRNA that folds to regulate transcription
Functions:
1) Inhibits transcription: riboflavin binds directly to transcript and it folds to form transcriptional terminator at front of operon
2) inhibits translation: mRNA secondary structure blocks ribosome from binding to SD sequence. Binding of MOLECULE allows translation
How do Riboswitches accomplish feedback inhibition
product of a pathway inhibits synthesis of enzymes that make it
Riboflavin produced and then it stops transcription
How do sRNAs work? What protein is required?
RNA chaperone Hfq binds to sRNAS. Hfq delivers sRNAs to target transcript. sRNAs interact with specific transcript and block SD sequence to inhibit translation
How does CsrA work
RNA binding protein, binds over the top of SD sequence, inhibits translation by blocking ribosomes.
Bacterial genomes generally consist of
single, closed circular chromosome
What are the monomers of DNA synthesis activated by?
phosphorylation
How is replication initiated? What sequence is required? What protein binds to that sequence? What is terC?
Origin is specific DNA sequence where replication begins (oriC) DnaA protein binds to oriC and recruits DNAP to begin replication.
ter C=terminus , DNA region where each replication fork ends during replication
What direction is DNA synthesized in? Which strand is continuous? Which strand is discontinuous?
DNA synthesized 5’-3’, DNAP III polymerizes DNA
Leading strand is continuous
Lagging strand is discontinuous
What direction does DNAP move from oriC? What is the name of this type of replicaton?
Bidirectional: sets off in opposite directions
Called theta replication because replicating chromosome is thought to look like Greek letter theta
What sequence is important for termination? What protein is required
ter sites
Protien Tus binds to ter sites and prevents anterograde replication
Tus stops DNAP near dif site
Why do the chromosomes need to be decatenated
After replication chromosomes are linked together like rings in a chain
What sequence is important for decatenation? What proteins are required?
dif site: recognized by proteins XerC/XerD that recruit DNA topoisomerase IV (cuts one chromosome at dif site, separates and then repairs)
Does DNAP stay positioned or does it move? What about the chromosomes?
DNP stays stationary at midcell, oriC moves toward poles
How does the cell deal with replication that is slower than cell divivion
Multiple replication forks: new rounds of replication begin before old round ends, each will inherit partially replicated genome.
At high growth rates, genes near origin have high copy #
Why are mutations in DNA more detrimental than mutations in RNA or errors in protein synthesis
DNA mutations alter all proteins encoded by that gene, alters all proteins of all descendants of mutant strain
What are the 3 causes/types of errors
Mismatch: DNAP makes mistake
Slipped Strand mispairing: DNAP tracks over repetitive sequence and slips forward or reverse
Mutagens: environmental factors that damage DNA
What is the most commonly occurring cause of errors in DNA? least common?
Mutagen: most common
Mismatch: least common
Why are mutagens heritable?
If unrepaired mutations are copied, and passed to next generation
Term for strain that has inherited a mutation
mutant
What is a genotype
DNA sequence of gene/chromosome
What is a phenotype
measurable/ observable trait conferred by a gene, mediated by proeins
What is an allele
version of a gene
What are the consequences of mutation
Change in gene sequence = change in protein sequence = change in protein function and shape
random mutation will almost always result in protein loss of function.
VERY rarely, gain of function mutation will occur that increases or changes protein activity
What is a missense mutation
Single base pair change that changes codon to a different AA
What is a nonsense mutation
single base pair change that changes codon to premature stop codon
What is a silent mutation
Change in codon, but codes for the same AA due to degenerativity. “silent” in protein sequence but may have a phenotype
What is a frameshift mutation
Insertion or deletion of base pairs in amounts not /3, completely alters subsequent AA sequence
What is polarity
Side effect of mutations within operons
