MCB 150 - unit 2

catabolic reactions

break down of larger molecules to smaller ones

often exergonic

anabolic/biosynthetic reactions

synthesis of larger molecules

often endergonic and coupled

energy intermediates

molecule formed when another is broken down

e.g covalent bonds in glucose are broken down and ATP forms with the released energy

substrate-level phosphorylation

when an enzyme directly transfers a phosphate from an organic molecule to ADP and making ATP

regulation

occurs within cells (e.g gene regulation), from signals from the environment (e.g cell signaling pathways), from enzymes (e.g biochemical), or the rate-limiting step

regulators stop pathways at the farthest back step that’s unique to that pathway

positive regulator

allosteric regulator that increases the activity of enzymes

negative regulator

allosteric regulator that decreases the activity of enzymes

feedback inhibition

type of noncompetitive inhibition

when the product of a metabolic pathway inhibits an enzyme that acts early in the pathway (ONLY inhibit the earliest step in the pathway for that specific product, don’t inhibit another pathway or else)

prevents overaccumulation of product

anaerobic respiration

cells use a substance that’s not O2 as the final electron acceptor in the ETC

e.g Escherichia coli produces nitrate reductase which produces nitrate as the final electron acceptor (which gets reduced to nitrite)

fermentation

produces ATP via substrate-level phosphorylation without oxygen

sometimes in anaerobic conditions only glycolysis is used to make ATP and NAD+ runs out and then glycolysis stops

usually in anaerobic conditions, pyruvate is instead reduced to lactate, using electrons from NADH, so NAD+ levels are increased (good)

once aerobic conditions presume, the lactate can be converted back to pyruvate or glucose

fermentation produces significantly less ATP, yielding only 2 ATP (vs 36 ATP)

glycolysis

glucose is broken into 2 pyruvate and a net energy yield of 2 ATP and 2 NADH

occurs in the cytosol in eukaryotes and prokaryotes, anaerobically and aerobically

endergonic until pyruvate are formed (then it is exergonic)

when 2 ATP is used, glucose is converted to fructose-1,6-biphosphate, and the free energy of glucose rises so later reactions can be exergonic

fructose-1,6-biphosphate then breaks into 2 glyceraldehyde-3-phosphate molecules

lastly, 4 ATP, 2 NADH, and 2 pyruvate form

kinase

a type of protein enzyme that transfers a phosphate group from a high-energy molecule (like ATP) to a specific substrate molecule (phosphorylation)

phosphofructokinase

an enzyme that regulates glycolysis by regulating the rate-limiting step

used between the conversion of fructose-6-phosphate to fructose-1,6-biphosphate (phosphorylates it)

cofactor

a non-protein chemical compound or metallic ion that an enzyme needs to act as a catalyst in a biochemical reaction

e.g inorganic compounds and coenzymes (ATP, NADH, FADH2)

intermembrane space

the narrow aqueous region situated between the outer membrane and the inner membrane in mitochrondria

because of porins, the composition of ions and small molecules in and outside of here is the same as the cytoplasm

pyruvate oxidation

the pyruvate from glycolysis is transported to the mitochondrial matrix

pyruvate is then oxidized where a CO2 and an electron is removed from pyruvate (technically it’s a carboxyl group (COOH), but it becomes CO2 & e- & H+), which generates energy to make a NADH and the remaining acetyl group is attached to coenzyme A to make acetyl CoA

pyruvate dehydrogenase

oxidizes pyruvate (by removing CO2 & e- & H+) during pyruvate oxidation

citric acid/krebs cycle

occurs in the mitochondrial matrix

the acetyl groups are removed from acetyl CoA and attached to oxaloacetate to form citrate and 2 CO2 + 3 NADH + 1 FADH2 + 1 GTP are released from each

after 8 steps, oxaloacetate is regenerated (cycle)

this cycle is inhibited when ATP and NADH levels are high, and stimulated when NAD+ and ADP levels are high

metabolic cycle

particular molecules enter/leave the cycle

there is no beginning or end

e.g krebs cycle

oxidative phosphorylation

the electrons from NADH and FADH2 are sent into the ETC

occurs in the cristae in eukaryotes and in the plasma membrane of prokaryotes

regulation occurs because of ATP (inhibits) and ADP (promotes) levels

the max number of ATP molecules that can be made during oxidative phosphorylation from one glucose molecule is 36, but this rarely occurs because some NADH and FADH2 are used for anabolic pathways and energy from the gradient may be used for other purposes (e.g the uptake of pyruvate into the mitochondrial matrix)

electron transport chain (ETC)

made of a group of protein complexes and small organic molecules in the inner mitochondrial membrane (in the plasma membrane of prokaryotes)

electrons are passed from one component to the next through redox reactions

each new component has a higher electronegativity (and lower energy) than the last, oxygen being the last one (where oxygen combines with protons to form water)

NADH donation in ETC

NADH first donates 2 electrons to NADH dehydrogenase (complex I), which transfers them to ubiquinone, cytochrome bc1 (complex III), cytochrome c, cytochrome oxidase (complex IV), then finally O2

FADH2 donation in ETC

FADH2 first donates 2 electrons to succinate dehydrogenase (complex II), then to ubiquinone, cytochrome bc1 (complex III), cytochrome c, cytochrome oxidase (complex IV), then finally O2

contributes less to the electrochemical gradiant (because it starts later + complex II doesn’t pump H+ across the membrane because it’s not a transmembrane complex), and so fewer protons are pumped across the membrane through FADH2 contribution

chemiosmosis

some of the energy that is released during the movement of electrons is used to pump H+ across the inner mitochondrial membrane to the intermembrane space, which creates an H+ electrochemical gradient or excess positive charge outside the matrix

as H+ flows back into the mitochondrial matrix from the membrane embedded portion of ATP synthase, the free energy is harnassed and ATP is made from ADP and Pi

ATP synthase

an enzyme that synthesizes ATP

ATP synthase can rotate clockwise when an H+ ion is bound to the membrane channel and rotates the ion into the matrix

ADP and Pi bind tightly to ATP synthase, which strains their chemical bonds, and ATP is made; ATP binds weakly to ATP synthase and is released

energy transforms from potential → kinetic → mechanical → chemical energy as protons flow through the membrane to make ATP

transformation

cells take up free external DNA from it’s environment and incorporates it into it’s own DNA

discovered by Griffith

pH of DNA and RNA

they are both acidic and release H+ in solution

DNA structure

a base is attached to the 1’ carbon and the phosphate group is attached to the 5’ position

DNA bonds by the 5’ end of a phosphate group attaching to the 3’ end on the current nucleic acid’s -OH

phosphoester linkage

connects the sugar molecule at 3’ to the phosphate group of another

a covalent bond between oxygen and phosphorus

phosphodiester linkage

viewing the connection between nucleic acids as a phosphate group connected to 2 sugars

backbone

sugars and phosphate groups linked together in DNA/RNA by phosphodiester bonds where the bases point inward

directionality

based on the orientation of sugar molecules

said to be going from 5’ to 3’ when the 5’ end has a phosphate group and 3’ has an OH group

abbreviated as 5’-[sequence]-3’

helical structure

Franklin and Wilkins found that DNA had 2 strands and was a helical structure (NOT that it was a double helix) through x-ray diffraction; also found that sugars and phosphates were on the outside and bases were on the inside, and always uniform in width

Watson and Crick proposed the structure of DNA and won the Nobel Prize (along with Wilkins); they found the double helix structure and pyrimadine-purine pairs (because the distance is fixed at 2 nm)

one turn of the double helix is 3.4 nm in length and compromises about 10 base pairs

chargaff’s rules

equal amounts of A & T, and G & C in all organisms

base pairs

A & T pair with 2 hydrogen bonds

G & C pair with 3 hydrogen bonds

antiparallel

one strand must run from 5’ to 3’ while the other runs from 3’ to 5’

grooves

indentations where the atoms of the base make contact with water

major grooves and larger than minor grooves

central dogma of molecular biology

the flow from DNA to RNA to proteins (from transcription to translation)

occurs in prokaryotes and eukaryotes

semiconservative mechanism

the new DNA strands in replication are half parent half daughter

conservative mechanism

the parent DNA strands are conserved together in replication

dispersive mechanism

segments of parent and daughter DNA are interspersed in both strands in replication

Meselson and Stahl

tested which mechanism of replication was most accurate using E coli and 14N + 15N

first grew all the bacteria in 15N then switched to 14N

since 15N sinks and 14N floats, and a mix of them remains in the middle, concentrations of each could be found

after 1 generation in 14N, all were half-heavy, and after 2 generations, both floating and half-heavy were observed so the semiconservative mechanism was proved to be the accurate one

origin of replication (ori)

site within a chromosome that serves as a starting signal for DNA replication

strands are separated at the ori and synthesis of new DNA will occur from both parent strands in both directions away from the ori

bacteria have one while eukaryotes have multiple

replication fork

the y-shaped region where DNA unwinds

bidirectional replication

the two replication forks move away from the origin in opposite directions during replication

in both prokaryotes and eukaryotes

lagging strand

made as a series of small fragments (okazaki) that later are connected

occurs in the opposite direction from the movement of the replication fork

okazaki fragments

fragments of the lagging strand

leading strand

made in the direction the fork is moving

synthesized as one long continuous molecule

DNA helicase

an enzyme that moves the separated strands at each fork outward from the origin

helicase binds to one of the DNA strands and travels from 5’ to 3’, using ATP to break hydrogen bonds

single-strand binding proteins

coats both DNA strands to prevent rebinding during replication (e.g after topoisomerase in prokaryotes)

DNA topoisomerase

removes and adds supercoils in DNA replication (e.g before replication forks in bacteria)

helps chromosomes fit in cells/nucleus by nicking, unwinding/winding, and resealing DNA; can relax and supercoil DNA when necessary

supercoils

knots that form when helicase moves through a DNA strand

DNA polymerase

enzyme responsible for covalently linking nucleotides together to form DNA strands

it breaks a bond between the first and second phosphate in deoxynucleoside triphosphates and attaches the resulting nucleotide with one phosphate group to the 3’ end of a growing strand via a phosphoester bond

the breakage of the covalent bond releases pyrophosphate (PPi) and energy to covalently connect adjacent nucleotides

if a DNA/RNA is already attached to a template strand, polymerase can elongate the given strand by making DNA in a 5’ to 3’ direction (but cannot synthesize DNA on a bare template strand)

deoxynucleoside triphosphates

polymerase helps these bind to the template strand

why is DNA replication very accurate

hydrogen bonding between complementary pairs is more stable than mismatched pairs

the active site of DNA polymerase is unlikely to catalyze bond formation between adjacent nucleotides if a mismatched pair is formed

DNA polymerase can identify mismatched nucleotides and remove it from the daughter strand

proofreading

when DNA polymerase identifies a mismatched pair it reverses it’s direction and digests links from 3’ to 5’ until the mismatched base, then it returns to normal direction

DNA polymerase III

after primase makes a RNA primer at the origin, DNA polymerase III attaches nucleotides in the 5’ to 3’ direction as it slides toward the opening of the replication fork

in the lagging strand each okazaki fragment requires it’s own primer, and polymerase III extends the primer to synthesize the remainder of the fragment

DNA polymerase III has 3’ to 5’ exonuclease activity, but cannot do it in the 5’ to 3’ direction

only in prokaryotes

has a clamp protein

can do proofreading

processivity

a characteristic of an enzyme that allows it to perform on a substrate without falling off

clamp protein

subunit of DNA polymerase III that allows the enzyme to slide along the template strand without falling off (grants processivity to polymerase III)

DNA polymerase I

removes RNA primers and replaces the empty regions with DNA to complete the synthesis of okazaki fragments

digests linkages between nucleotides in each RNA primer from 5’ to 3’ (exonuclease)

found only in bacteria

can do proofreading

DNA primase

makes a complementary primer that is actually a short segment of RNA that prime the process of DNA replication (so polymerase can add to it)

DNA ligase

catalyzes the formation of a covalent bond between the first and last nucleotides in DNA and joins okazaki fragments together

nuclease

an enzyme that hydrolyzes a phosphodiester linkage (e.g digests linker DNA in chromatin)

exonuclease

a nucleaese that hydrolyzes nucleic acid from the end of a chain (e.g proofreading)

endonuclease

a nuclease that hydrolyzes nucleic acid internally

chromosomes

contains the genetic material

made of proteins and nucleic acids (chromatin)

DNA, not proteins, was found to hold the genetic information in chromosomes, originally thought it was proteins because it’s 20 amino acids vs 4 nucleotides (proved by Avery)

prokaryotes have circular chromosomes, eukaryotes have linear structure for their genome but they can also be circular when not in the nucleus

chromatin

looks like beads on a string, beads are nucleosomes, string is nucleic acids; connected ionically

histones

small, basic proteins that are basically the same between species

bacteria don’t have histones (but has histone-like proteins), but archaea do

major types are H1, H2A, H2B, H3, H4

H1 is the only histone not in the bead, it’s around the bead and is like tape to keep the chromatin together

there is only 1 H1 per nucleosome, but 2 of each of the others in the center

chromatosome

nucleosome + H1 + a bit more

nucleosome

the basic repeating structural unit of chromatin that packages eukaryotic DNA into a compact shape inside the cell nucleus

~10nm in size, made of 8 histones + ~146 base pairs of DNA

telomere

region at the ends of eukaryotic chromosomes that has a specialized form of DNA replication

has a repeated sequence (e.g 5’-TTAGGG-3’ in humans)

‘3 overhand

region at the 3’ end of the telomere with no complementary strand

since there’s no place for a primer, it cannot be replicated (chromosomes will shorten over time)

telomerase

prevents chromosome shortening by attacking many copies of a DNA repeat sequence at the ends of chromosomes

contains both protein and RNA (has it’s own template)

the RNA portion is complementary to the DNA repeat sequence, which allows telomerase to bind to the 3’ overhang

euchromatin

a type of interphase chromatin

loosely condensed, distributed throughout the nucleus, most are 30nm fibers

heterochromatin

a type of interphase chromatin

highly condensed, found mostly at the periphery of the nucleus

transcription

information from genes is copied into an RNA intermediate/transcript

occurs in the nucleus in eukaryotes and the cytoplasm of prokaryotes

always occurs in the 5’ to 3’ direction as the template strand is always read from 3’ to 5’

mRNA

carries information from DNA to ribosomes

amplification

most genes are present in 1 or 2 copies in a cell (very little template for making proteins) so amplification is the process of multiplying proteins

protein-coding genes

produces an RNA molecule that contains the information to specify a polypeptide with a particular amino acid sequence

promoter

contains when and where transcription begins (based on the positioning of the promoter, it determines whether the bottom or top strand is used as a template during transcription)

terminator

a DNA sequence that signals the end of transcription (signals RNA polymerase to release the RNA transcript)

not an immediate stop but takes time

-10 site

around 10 base pairs upstream from the start

has a consensus sequence of TATAAT on the coding strand (read from 5’ to 3’)

the ending -AT are the most important to be present

the closer it is to the consensus/conserved sequence, the higher chance RNA polymerase will recognize and bind to the site

-35 site

around 35 base pairs upstream from the start

has a consensus sequence of TTGACA on the coding strand (read from 5’ to 3’)

the beginning TT- are most important to be present

the closer it is to the consensus/conserved sequence, the higher chance RNA polymerase will recognize and bind to the site

consensus sequence

consistent sequence with specific pattern

conserved sequence

consistent sequence always in nearly the same spot

regulatory sequences

sites for the binding of regulatory proteins on protein-coding genes

can increase or decrease transcription rate

RNA polymerase (eukaryotes)

eukaryotes’ transcription involves more proteins than bacteria

eukaryotes have 3 types of RNA polymerase: RNA polymerase I transcribes rRNA, RNA polymerase II transcribes mRNA, RNA polymerase III transcribes tRNA (and some rRNA)

each type of RNA polymerase in eukaryotes recognizes it’s own promoter regions, which is why they only make specific RNAs

RNA polymerase (prokaryotes)

bacteria only has one type of RNA polymerase, but it usually has more than one type of sigma factors

transcription factors

instead of sigma factors, eukaryotes require 5 transcription factors to initiate transcription

there are proteins that influence the ability of RNA polymerase to transcribe genes

preinitiation complex

RNA polymerase II binds to the promoter

polymerase II and the 5 transcription factors form this complex to unwind DNA to initiate transcription

holoenzyme

consists of 6 subunits (2 alpha, beta, beta’, omega, sigma)

core enzyme

holoenzyme without the sigma subunit/factor

sigma factor

in prokaryotes it binds to RNA polymerase and then helps bind it to a promoter

RNA polymerase

synthesizes an RNA strand by copying a DNA template during transcription

open complex

formed when the DNA strands are separated near the promoter by the holoenzyme during initiation

elongation (transcription)

RNA polymerase synthesizes the RNA transcript now

sigma factor is release and RNA polymerase slides across the DNA and maintains an open complex

RNA is transcribed here, then after DNA is rewinded into a double helix

termination (transcription)

the RNA transcript dissociates from the DNA

termination occurs on per gene basis where each gene can have either way of terminating in an organism, but always terminates that way

rho-dependent termination

rho binds to RNA at rut sites and uses ATP to catch up to RNA polymerase near the 3’ end/transcription complex

acts as DNA helicase and can peal away the RNA from the DNA and makes the RNA polymerase deattach

rut sites determine whether termination occurs with rho or intrinsically

intrinsic termination

an end of RNA with a hairpin loop because of a self-complementary sequence (e.g full of guanines then full of cytosines)

the bulky structure of the hairpin make it so RNA polymerase can’t access the hairpin well and a weak trail of A-U bonds (uracil on the RNA) at the end of the hairpin make it susceptible to get hit away from the DNA

splicing

introns are removed and exons are connected

most common in multicellular eukaryotes, but occurs in prokaryotes rarely

occurs post-transcriptionally but also acts co-transcriptionally