Download Microbes in nutrition Digestion vast majority of GI tract bacteria are

I. Microbes in nutrition
A. Digestion
1. vast majority of GI tract bacteria are obligate anaerobes
2. anaerobic degradation requires several groups of bacteria working together
a. energy is shared between groups
b. final (terminal) metabolic group uses hydrogen to make a reduced endproduct
(1) methanogens make methane
(2) acetogens make acetate
3. fatty acids produced by fermentative pathways provide energy for animal
B. Herbivores
1. ruminants perform extensive fermentation in the rumen
a. cellulose hydrolyzed to glucose
b. glucose fermented to fatty acids (butyrate, propionate, acetate, succinate, etc.),
carbon dioxide, and hydrogen
c. methanogens reduce carbon dioxide with hydrogen to make methane
2. termites perform a similar operation in their hindgut
II. Food production
A. lactic acid bacteria (LAB) preserve food by lowering the pH
1. strictly fermentative, aerotolerant anaerobes
2. vegetables (sauerkraut, kim chee, etc.)
3. dairy (yogurt, cheese, kefir, etc.)
4. meat (summer sausage, salami)
5. grain (sourdough starter)
6. probiotics
B. Saccharomyces cerevisiae
1. ferments glucose to ethanol plus carbon dioxide
a. doesn’t hydrolyze starch
b. facultative anaerobe
2. used to make bread and fermented beverages (e.g., wine, beer)
a. grapes contain sugar and can be directly fermented
b. grains (e.g., barley) contain starch that must be hydrolyzed to glucose
3. starch is converted to glucose by amylase
a. germinating seeds (malting)
b. saliva
4. other yeasts (e.g., Pichia pastoris) can be used as single cell protein (SCP)
C. Many other foods are made or modified by microbes
1. soy sauce (Aspergillus oryzae)
2. aged meat
3. vinegar (Acetobacter acetii)
4. chocolate (yeasts, LAB, acetic acid bacteria)
5. tempe (Rhizopus)
D. Food spoilage
1. transformation can be detrimental
a. tastes ruined
b. disease transmission
c. toxins
2. environmental factors influence the ease of food spoilage
a. pH influences microbial population
(1) low pH favors growth of yeasts and molds
(2) at neutral or alkaline pH, bacteria are more dominant
(3) putrefaction = anaerobic breakdown of proteins, releasing foul-smelling
amine compounds
(4) common with meats
b. water availability (dehydration) can be used to preserve food
c. oxidation-reduction potential influences spoilage
(1) cooked meat or broths have low redox potentials, ideal for growth of clostridia
(2) anaerobes grow for long periods in sealed containers
d. physical structure influences spoilage
(1) sausage and hamburger have increased surface area and grinding spreads
bacteria throughout the food
(2) fruits and vegetables have protective skins, although spoilage microorganisms
have enzymes that allow penetration of peels and rinds
e. many foods contain natural antimicrobials
(1) aldehydes and phenolics in cinnamon, mustard, and oregano
(2) coumarins in fruits and vegetables
(3) lysozyme in cow's milk and eggs
f. plastic films (shrink-wrapped foods) allows penetration of oxygen
(1) increased surface growth of bacteria
(2) excess CO2 lowers pH, selects for lactobacilli
3. approaches to food preservation
a. physical removal (filtered beer)
b. temperature
(1) refrigeration retards microbial growth
(a) selects for psychrophiles and psychrotrophs
(b) microbial growth reported at -10oC in fruit juice concentrates, ice cream,
some fruits
(2) pasteurization reduces microbial populations
(3) canning sterilizes food
c. chemical additives are used as preservatives
(1) organic acids, sulfite, ethylene oxide, sodium nitrite, ethyl formate
(2) organic acids work best at low pH (undissociated)
(3) nitrites prevent germination of clostridial spores
(a) preserve red color in meat
(b) can form carcinogenic nitrosamines
d. gamma irradiation works well with moist foods because the process produces
peroxides in cells, disrupting cellular components
4. Food poisoning
a. food-borne infection = ingestion of pathogen, followed by growth accompanied
by tissue invasion and/or release of toxins in the host intestine
III. Applied microbial ecology and diversity
A. Environmental microbiology
1. nearly all chemical changes that take place on earth are caused by biological systems
a. non-biological agents
(1) volcanoes (alter rocks and atmosphere)
(2) lightning (N-oxides, ozone)
(3) UV light (ozone)
(4) rain (erosion, solutions)
(5) radioactivity (heat at core, changes in neighboring rocks)
b. biosphere = dynamic system of chemical changes brought about by biological
agents at the expense of solar energy
(1) chemical changes brought about by one biological agent are reversed by some
other biological or non-biological activity
(2) elements undergo cyclic changes, from organic to inorganic and back to
organic (C, N, O, S, metals)
2. microorganisms are essential components of every ecosystem
a. ecosystem = community of organisms and their physical and chemical
environment that functions as an ecological unit
b. primary production = synthesis of new organic material from CO2 and other
inorganics
c. decomposition = breakdown of accumulated organic matter
Tertiary-level consumers
Secondary-level consumers
Primary consumers
Bacteria and Fungi
Primary Producers
d. primary producers accumulate organic matter
(1) terrestrial = vascular plants
(2) aquatic = cyanobacteria and algae
e. decomposers mineralize organic matter to simpler, inorganic matter
3. microbial functions in natural environments
a. mineralization of organics
b. serve as nutrient-rich food source for other chemoheterotrophic microorganisms
c. serve as food source for protozoa, nematodes, and soil insects, thus creating a
food web (network of interlinked food chains)
d. modifying substances for use by other organisms
e. changing amounts of materials in soluble and gaseous forms
f. producing inhibitory compounds that decrease microbial activity or limit survival
and functioning of plants and animals
4. destructive effects (human viewpoint)
a. general food spoilage
b. corrosion
(1) sulfides (phosphides)
(a) metals
(b) concrete (thiobacilli)
i) buildings
ii) sewage pipelines
(2) cathodic depolarization
c. oil souring
(1) oil/water interface
(2) hydrocarbon degradation?
d. water pollution
(1) anaerobiosis in stagnant water
(a) kills O2-requiring life
(b) horrible odors
(c) self-perpetuating
(d) canals of Venice (high sulfide)
(2) artificial pollution can upset natural balance
(a) whole lake can turn red with photosynthetic purple sulfur bacteria
(b) ". . . and all the waters that were in the river were turned to blood. And all
the fish that was in the river died; and the river stank, and the Egyptians
could not drink of the water of the river . . ." (Exodus 7:20-21)
e. cellulolytic organisms
f. pathogens
(1) most diseases are caused by microbes
(2) biologically, disease is important because it limits populations
(3) major stimulant to development of microbiology as a science
5. beneficial aspects
a. remove detritus (dead organic material)
b. sewage treatment
(1) settling tanks (sludge settles)
(a) liquid goes to aerobic ponds (cells + CO2)
(b) cells (activated sludge) added to settled sludge
(2) sludge treated in anaerobic digestors
(a) methanogenic ecosystem (CO2 + CH4)
(b) treated, dewatered sludge is dumped (could be used as fertilizer, but often
low in N, P, S)
c. industrial waste treatment
(1) sugar/molasses processing
(2) paper industry
d. petroleum wastes (spills, sea oils)
(1) detergents treat symptoms, but no real removal (often act as disinfectants,
killing bacteria)
(2) hydrophobic fertilizers (N, P)
e. bioremediation of xenobiotics (e.g., pesticides, plastics)
B. Geomicrobiology
1. microbial community has a major role in biogeochemical cycling
a. involves biological and chemical reactions
b. often involves oxidation-reduction reactions
c. microorganisms essential for C, N, S, and Fe cycles
2. carbon can be in reduced forms (CH4) or oxidized forms (CO, CO2)
a. reductants (H2) and oxidants (O2) influence course of biological and chemical
reactions
(1) H2 commonly produced anaerobically, along with CH4
(2) aerobically, the H2 and CH4 can be oxidized
b. methane levels in air increasing approx. 1% each year
(1) ruminants + manure lagoons
(2) coal mines
(3) sewage treatment plants
(4) landfills
(5) marshes
(6) rice paddies
c. carbon fixation performed by photo- and chemo-autotrophs
(1) cyanobacteria and green algae
(2) photosynthetic bacteria (Chromatium, Chlorobium)
(3) aerobic chemolithoautotrophs
3. Nitrogen cycle
a. nitrogen (N2) gas most abundant component of atmosphere (79%)
(1) largely biologically unavailable
(2) about 0.03% is combined (fixed) as nitrates (NO3), nitrites (NO2), ammonium
(NH4), organic compounds (proteins and nucleic acids)
b. fairly complex, more of a web than a cycle
(1) higher plants use NO3 and NH4
(2) animals use organic forms
(3) microbes use NO2, NO3, NH4, N2 and organics
(4) cycle has 4 phases: nitrogen fixation, ammonification, nitrification, and
denitrification
c. nitrogen fixation
(1) nitrogen fixation performed only by bacteria
(a) 1st step in synthesis of virtually all nitrogenous compounds
(b) unique enzyme system that anaerobically reduces N2 to forms that can be
used by most organisms
(c) primary product is ammonium ion, NH4+
(2) N2-fixers can be free-living or symbiotic with plants
(a) free-living: Azotobacter, Azospirillum, some clostridia, cyanobacteria
(Anabaena, Nostoc)
(b) symbiotes: rhizobia (Rhizobium, Bradyrhizobium, Azorhizobium) and
legumes (soybeans, peas, alfalfa, clover)
i) form root nodules
ii) bacteria provide reduced nitrogen to plant
iii) plant furnishes nutrients and energy to bacteria
d. ammonification
(1) decomposition of nitrogen-containing organics produces NH4+ (Clostridium,
Proteus)
(2) NH4+ can be used by some plants, or acted upon by other groups of bacteria
e. nitrification
(1) oxidation of ammonium to NO2 and NO3
(2) makes nitrogen available in another useful form
(3) two phases: NH4+  NO2, NO2  NO3
(4) nitrates used by variety of organisms
f. denitrification
(1) NO3  NO2  NO  N2O  N2
(2) essentially the reverse of nitrification
4. sulfur cycle
a. similar to nitrogen cycle, but drawn from sedimentary deposits instead of the
atmosphere
b. exists in elemental form (S), hydrogen sulfide gas (H2S), sulfate (SO4), and
thiosulfate (S2O3)
c. most oxidation-reductions performed by bacteria
d. plants and some microbes require SO4
e. animals must have organic source
(1) cystine, cysteine, methionine
(2) sulfide bonds contribute to stability and configuration of proteins
5. sulfur deposits
a. occur in confined deposits called domes
(1) always near calcium sulfate deposits
(2) usually close to oil deposits
b. probably formed by intense microbial activity during geological era of warmth
and sunshine, probably while a sea was drying
(1) SRB used calcium sulfate to make calcium sulfide
(2) sulfide oxidized to calcium carbonate and free sulfur, probably through action
of photosynthetic sulfur bacteria
(3) lack of air probably prevented oxidation of the sulfur
c. calcium sulfate crystallized out, sulfur sedimented, organic matter may have
contributed to oil formation
(1) process appears to be occurring today
(a) lakes in Libya rich in calcium sulfate produce tons of sulfur each year
(b) SRB reduce sulfate to sulfide at expense of organic matter formed by
colored sulfur bacteria
(c) colored sulfur bacteria make organic matter from CO2 using sunlight or
sulfide, also producing large amounts of elemental sulfur
(2) other evidence is that during biological sulfide formation, some separation of
natural sulfur isotopes occurs, but not during chemical sulfate reduction
(a) chemically deposited sulfur have identical isotope ratios
(b) biologically deposited sulfur are richer in the lightweight isotope (residual
sulfate richer in heavier isotope)
6. coal deposits
a. huge forests of plants (Carboniferous era); warm, humid environment; dying
vegetation formed vast compost heap
b. anaerobic fermentation took place with production of methane and humic acids
(1) humic acids are related to phenols
(2) prevent additional microbial activity
(3) reason why organics in peat bogs so well preserved
c. peat is plant material that is depleted in oxygen
(1) under pressure (sand and rocks) peat is compressed to coal
(2) lignite (structurally like peat)  bituminous  anthracite
7. oil formation
a. not known (accepted) if microbial origin
(1) not reproducible in laboratory
(2) oil deposits have characteristics that make biological origin likely
b. oil deposits rich in anaerobic bacteria
(1) particularly SRB
(2) cultures that produce oil-like compounds are mixed cultures including SRB
c. crude oil often contains porphyrins (chemicals derived from respiratory enzymes,
not known to occur abiotically)
d. certain hydrocarbons are optically active (known to occur only as a result of
biological systems)
e. data not conclusive - all features could have resulted from microbial activity
after oil was formed
C. Applied microbiology
1. the properties of specific microorganisms and ecosystems can be exploited for
practical application
a. industrial microbiology = use of microorganisms to produce organic chemicals
(solvents, feedstocks), enzymes, antibiotics, hormones, steroids, and food
supplements
b. other applications = sewage treatment, control of insects, recovery of metals,
various environmental uses
c. biotechnology = generally implies use of recombinant DNA techniques to design
proteins or modify gene expression
(1) originally strains were selected and improved crudely, at most using nonspecific mutagenesis
(2) modern methods allow recombination using markedly different organisms
2. industrial fermentation = large scale cultivation of microorganisms
a. primary metabolite = produced during growth phase and normally involved in
synthesis of new cells
(1) amino acids
(2) nucleotides
(3) fermentation endproducts
(4) enzymes
(5) cofactors
b. secondary metabolite = usually accumulate after the active growth phase and
have no direct relationship to synthesis of cell material or normal growth
(1) antibiotics
(2) mycotoxins
3. recombinant DNA technology
a. strains often include biological markers to allow easy identification of mutants
(1) nutritional requirements
(2) antibiotic resistance
b. protein engineering = modification of a protein molecule to enhance functioning
genes created by site-directed mutagenesis or by insertion of chemically
synthesized DNA
c. metabolic engineering = restructuring of metabolic networks by introduction of
proteins from other cells
(1) allows synthesis of entirely new products and intermediates
(2) products include synthetic medical peptides that promote wound healing and
blood coagulation, treat cancer or AIDS, influence sexual dysfunctions, help
hormonal disorders
(3) major advantage of biological synthesis is production of specific
stereoisomers (e.g., thalidomide)
4. biocontrol of pests
a. Bacillus thuringiensis produces an intracellular protein toxin crystal that acts as
an insecticide
(1) crystal is solubilized only by insects with high pH in midgut
(2) fermentor technology allows accumulation of toxin for solubilization prior to
large-scale distribution
(3) current efforts directed towards production of toxin using plasmid-coded
genes
b. viruses that are pathogenic for specific insects (usually butterflies and moths)
have been produced commercially
5. biopolymers (gums) have been produced and used as additives in pharmaceuticals
and foods (stabilization, water retention, flow characteristics, film-forming)
a. production not subject to climatic, political, or economic constraints
b. production facilities can be located near sources of inexpensive substrates (or
endusers)
6. biosensors can use living microorganisms or active enzymes to measure specific
components
a. enzyme (or metabolic) activity used to generate electrical impulse
b. impulse related to presence of specific compounds, even in highly mixed or
"dirty" environments
c. replacing and expanding applications of bioassays
(1) food components (alcohol)
(2) pollutants
(3) toxic gases
(4) direct measurement of flavors, essences, and pheromones
7. biodeterioration
a. fuels
(1) usually occurs at water/hydrocarbon interface
(2) can be detrimental
(a) oil souring
(b) jet fuel deterioration by Cladosporium resinae
(3) can be applied to bioremediation of oil spills
(a) 1st form of bacterial life patented (Pseudomonas)
(b) oleophilic fertilizers
(c) application of specific organisms or communities
b. paper cellulosic materials
(1) microbes can lower process efficiency by growing in treatment solutions
(2) mercury compounds often used as biocides, being replaced by chlorine,
phenols, and organosulfides
(3) waste streams can be bioprocessed for cleanup
c. metals
(1) corrosion, especially anaerobically, is a major problem
(2) Thiobacillus ferrooxidans can leach up to 70% of copper content from lowgrade ores
d. textiles and leather
(1) common in high humidity or tropical areas
(2) controlled with phenols and copper compounds
e. paints
(1) control methods use to include addition of mercury compounds
(2) currently use copper compounds (especially marine applications) and
quartenary amines
IV. Industrial microbiology
A. Three primary areas
1. produce cells as the desired product
a. yeast for food
b. cells to be used for other processes
(1) biological warfare
(2) bioremediation
2. use cells to carry out a process
a. modify molecules from one form to another
b. bioremediation (breakdown of pollutants)
3. microbial products
a. alcohol
b. antibiotics
c. enzymes
B. Anaerobic degradation
1. complex ecosystem
a. unlike aerobic degradation, involves several metabolic groups of anaerobes
b. hydrolytic bacteria break down macromolecules
c. fermentative bacteria produce organic acids
d. acetogens produce acetic acid and hydrogen
e. final group reduces an anaerobic electron acceptor
(1) methanogens reduce CO2
(a) produce CH4
(b) common in GI tracts, freshwater systems
(2) sulfate-reducing bacteria more common in marine systems
(a) outcompete methanogens
(b) reduce sulfate (SO4) to hydrogen sulfide (H2S)
2. important natural and applied process
a. breakdown of materials in soils, sediments, GI tracts
b. important for waste treatment
C. wastewater treatment
1. water quality became important with onset of epidemics spread by waterborne
pathogens
a. cholera
b. typhoid fever
c. dysentery
2. waste treatment and water purification largely eliminated waterborne epidemics, but
left the problem of waters overloaded with sewage
a. health
b. esthetics
c. environmental
3. problems intensified with increasing populations and additions of xenobiotics
(compounds that don’t occur in nature)
4. BOD = indirect measure of organic matter in aqueous environment
a. measure of oxygen consumption required for microbial oxidation of degradable
organics
b. for wastes, a high value is bad
c. often used as a measure in determining organic carbon in environmental samples
5. chemical oxygen demand (COD) = amount of chemical oxidation required to
convert organic compounds in water and waste water to CO2
a. oxidation performed chemically (permanganate consumption) or by heating (CO2
evolved)
b. gives total organic carbon (TOC)
6. BOD lower than COD due to microbial biomass and recalcitrant (hard to degrade)
chemicals
7. sewage overload in natural waters lowers or exhausts the dissolved oxygen (DO),
slowing microbial processes drastically
a. oxygen is not replenished rapidly enough and anaerobic conditions result
(1) aerobic organisms die, increasing the oxygen demand
(2) anaerobic reactions (fermentation and respiration) give rise to noxious odors,
tastes, and colors
(3) turbidity and H2S production inhibit photosynthetic oxygen regeneration,
slowing the recovery process
b. biological diversity drops
8. Sewage treatment aims to reduce BOD prior to discharge
a. typically occurs in three stages
b. 1o = physical removal of larger objects, grit, floating scum, and grease using
screens, traps, or skimming devices
(1) occurs in settling tanks where solids are drawn off the bottom
(2) solids may be composted or digested anaerobically prior to disposal in a
landfill or as soil conditioner
(3) liquid portion subjected to further treatment or discharged if BOD lowere at
least 70%
(4) typically total BOD (solids and liquids) decreased only about 30-40%
c. 2o = microbial reduction of BOD, either aerobic or anaerobic
(1) original BOD lowered at least 80%
(2) effluent should have BOD less than 20 mg/l
(3) removal of nonbiodegradable organics and minerals, especially N and P salts
d. 3o = disinfection before release
(1) chlorination
(2) ozonation
(3) UV lights
9. Aerobic treatments
a. trickling filter
(1) sewage distributed by boom-type sprinkler over a bed of porous material
(stones)
(2) percolates through matrix, over aerobic biofilm
(a) biofilm contains bacteria, fungi, protozoa, nematodes, and rotifers
(b) absorb and mineralized dissolved organics
(c) passive aeration provided by porous nature of bed
(3) sloughed-off biomass allowed to settle, further clarifying effluent
(4) drawback of this inexpensive system:
(a) nutrient overload may lead to overproduction of slime, reducing aeration
and percolation
(b) cold winter temperatures severely reduce effectiveness of outdoor
facilities
b. rotating biological contractor or biodisc system
(1) closely spaced disks (usually plastic) are rotated in trough containing sewage
effluent
(a) disks coated with biofilm
(b) continuous rotation aerates
(c) overproduced slime sloughed off, can be settled out
(2) require less space than trickling filters, are more efficient and stable, produce
no aerosols
(3) higher initial investment
c. activated sludge
(1) open tank with stirring and air pumps
(2) high rate of nutrient utilization
(3) produces a lot of cell material
(4) more expensive than trickling filter or biodisc
D. Anaerobic treatments
1. generally slower (reduced metabolic rates), but doesn't require expense of aeration
2. can recoup expenses by recovering energy in methane (biogas)
3. anaerobic digestors (large-scale) are used for additional processing of sewage sludge
produced by primary and secondary treatments
a. economics not suitable for direct treatment of sewage (compared to aerobic)
b. primary used for processing settled sewage sludge and industrial wastes with high
BOD
4. conventional anaerobic digestors are large fermentation tanks designed for continuous
operation (culture)
a. mixing, heating, gas collection, sludge addition, and removal of stabilized sludge
all incorported in design of tanks
5. anaerobic digestion is usually represented as a 3- or 4-stage process
a. initial digestion of macromolecules by extracellular polysaccharidases, proteases,
and lipases to soluble materials
b. fermentation of the soluble materials to fatty acids, H2, and CO2
c. fermentation of fatty acids to acetate, H2, and CO2
d. methanogenesis from acetate or H2 plus CO2
V. Molecular biology and genetics
A. The Central Dogma of Biology describes how information (DNA) is used by cells to
make the machinery of life (enzymes)
1. coined by Francis Crick
2. essentially correct from conception except for RNA-directed DNA polymerase
(reverse transcriptase)
Replication
3.
B. Structure of DNA
1. purine or pyrimidine base + ribose or deoxyribose = nucleoside or deoxynucleoside
2. nuclecoside or deoxynucleoside + phosphoric acid = nucleotide or deoxynucleotide
3. DNA usually in double helix
a. anti-parallel strands joined by H-bonds between bases
(1) bases on opposite strands are complementary
(2) GC, A=T
4. gigantic nucleic acid molecule with two polynucleotide strands arranged in a double
helix
a. nucleic acid (RNA or DNA) = polymers of nucleotides
b. nucleotides = nitrogen base + pentose sugar + phosphate
c. nitrogen base = purines (2 rings) or pyrimidines (one ring)
(1) purines = adenine (A) or guanine (G)
(2) pyrimidines = thymine (T), cytosine (C) or uracil (U)
5'
HOC H 2 O
3'
HOC H 2 O
OH
H H
H H
H
H
OH H
H
H
OH O H
Ribose
Deoxyribo se
H
H
O
N
N
N
N
N
H
H
H
N
H
N
H
N
H
Ade nine
N H
N
H
Guan in e
H
H
O
N
O
H 3C
H
H
H
N
H
Thymine
H
N
N
N
H
OH
O
H
N
H
Cyt osine
O
H
N
O
H
Urac il
5. The DNA molecule consists of a deoxyribose sugar -phosphate backbone attached to
nitrogenous base crosspieces
a. phosphate substitution on 5' carbon on deoxyribose joins to 3' hydroxyl group on
neighbor 3'5'
b. bases attached to 1' C of deoxyribose
c. hydrogen bonds between anti-parallel strands join the two helices
(1) G-C, 3 bonds
(2) A-T, 2 bonds
6. Significance of DNA structure
a. maintenance of code during replication
(1) base pairing helps retain base sequence
(2) each strand provides a template
b. provides variety, since order of bases constitutes the genetic code
(1) for DNA 1000 bases long, combinations = 41000
7. RNA usually single stranded (can coil back on itself to form hairpins)
8. In cells, DNA tends to be supercoiled
C. Replication
1. replication = synthesis of DNA
2. DNA helix unravels and actual replication occurs at the replication fork
3. replication of bacterial chromosome begins at a single point, the origin
a. bidirectional, replicons (portion of genome containing an origin and replicated as
a unit) separate when forks meet opposite the origin
b. replication fork and associated enzymes may be attached to plasma membrane
4. eucaryotic DNA is linear and much longer than procaryotic DNA
a. many replication forks simultaneously
b. several origin sites along the DNA
5. DNA polymerases (3 different types) catalyze DNA synthesis 5'  3'
a. require dNTP, template
b. pol III main enzyme, some pol I
c. pol I and pol II probably mostly for repair
6. helicases unwind DNA (use ATP for energy)
7. single-stranded DNA binding proteins (SSBs) maintain strand separation
8. topoisomerases relieve supercoiling due to unwinding
9. replication of the leading strand (unwinding 3'  5') is continuous
10. replication of the lagging strand is discontinuous
a. small (Okazaki) fragments synthesized 5'  3' (1000-2000 nucleotides in
procaryotes, 100 in eucaryotes)
b. pol I removes RNA primer, synthesizes complementary DNA
c. DNA ligase links the fragments
(1) bacteria use pyrophosphate bond of NAD+ for energy
(2) other ligases use ATP instead
11. pol III has proofreading ability where wrong bases are excised and correct ones
inserted during replication
D. Transcription
1. transcription = synthesis of RNA under direction of DNA
a. RNA has sequence complementary to DNA template
b. uracil replaces thymine
c. mRNA = contains message for protein synthesis
d. tRNA = carries amino acids during protein synthesis
e. rRNA = ribosome components
2. mRNA is single stranded RNA containing directions for protein synthesis
3. RNA polymerase synthesizes RNA
a. requires NTP, DNA template
b. 5'  3'
c. copies only the sense strand of DNA
(1) different genes may be encoded on opposite strands
(2) gene = DNA segment or sequence that codes for a polypeptide, an rRNA, or
a tRNA (in other words, a functional product)
4. promoter = 6 base sequence, about 35 base pairs before transcription start point
a. Pribnow box (TATAAT) about 10 bases upstream of transcript
b. RNA pol recognizes, binds to promoter, and unwinds short DNA segment
around Pribnow box
5. terminators = stop transcription
a. all contain sequence that forms a hairpin loop (H2-bonding) that stops or slows
RNA polymerase
b. two types of stop signals or terminators
(1) UUUUUU after hairpin causes polymerase to stop and release mRNA
without any accessory factors
(2) rho factor () (no UUUUUU region)
(a) rho binds to mRNA, moves until it reaches paused RNA polymerase
(b) causes polymerase to dissociate from mRNA, probably by unwinding
mRNA-DNA complex
6. synthesis of rRNA and tRNA similar to mRNA except
a. molecules terminated by 5'-monophosphate rather than triphosphate found at
end of all primary transcripts (mRNA)
b. rRNA and tRNA are smaller than primary transcripts
c. tRNA contains bases other than A, G, C, U not in original transcript
d. primary transcripts undergo posttranscriptional modification or RNA
processing
7. In eucaryotes 3 RNA polymerases instead of 1
a. different promoters
b. heterogeneous nuclear RNA (hnRNA) = large RNA precursors, about 5000 50000 nucleotides long
c. posttranscriptional modification = hnRNA cleaved to final mRNA
d. RNA splicing removes introns from initial RNA transcript
(1) small nuclear RNA (snRNA) binds to splice junctions
(2) splicing of pre-mRNA occurs in large complex called a splicosome
e. ribozyme = self-splicing pre-rRNA molecules
f. rRNA and tRNA result from posttranscriptional processing
E. Translation
1. mRNA nucleotide sequence is translated into an amino acid sequence
a. the genetic code associates the three-nucleotide codon with the amino acid it
codes for
b. three codons do not code for an amino acid and are called stop or nonsense
codons
c. some amino acids have more than one codon = code degeneracy
d. each codon only codes for a single amino acid = code stringency
2. protein synthesis occurs on the ribosomes
a. in prokaryotes, mRNA often complexed with several ribosomes at once,
synthesizing several copies at once (polyribosome)
b. ribosomes can attach to mRNA being synthesized, so transcription and
translation can occur simultaneously (in eucaryotes, transcription and
translation machinery separated by nuclear membrane)
3. prokaryotic ribosomes are 70S organelles composed of a 50S and 30S
subunits,eucaryote ribosomes are 80S (60S + 40S)
4. ribosomes have three binding sites: A, P, and E
a. A site is where incoming tRNA with the next amino acid binds
b. P site is where the tRNA with the growing peptide chain binds
c. E site is where the “empty” tRNA is released from the ribosome
5. sequence of peptide synthesis:
a. peptidyl-tRNA moves from A site to P site
b. ribosome moves one codon along mRNA so that new codon is positioned at the
A site
c. next amino acid in the sequence, attached to the proper tRNA, binds to the A
site
d. the peptide chain attached to the tRNA in the P site attaches to the amino acid
in the A site
e. the ribosome moves along the mRNA so that the A site is empty (ready for next
amino acid), the P site has the tRNA with the growing peptide attached, the free
tRNA (formerly bound to the peptide) is in the E site and ejected
6.
7.
protein synthesis stops when ribosome reaches a nonsense or stop codon
a. release factors aid recognition of these codons
b. once stopped, peptidyl transferase hydrolyzes the peptide free from its tRNA
and the empty tRNA is released
c. ribosome dissociates from mRNA and separates into 30S and 50S subunits
as polypeptide leaves the ribosome, it begins folding into its final shape