MCB Exam 2 Lecture Notes

NAD+ accepts 2e and 1H+ to form NADH (other H+

floats around)

FAD accepts both 2e and 2H+ to form FADH2

Bond in NAD+ or NADH connecting the phosphate

groups is NOT a phosphodiester bond

In ATP, phosphate groups are unstable meaning that

since they are so close to each other, have positive

potential energy

Leftover energy in any coupled reaction with ATP is lost

as heat

Glycolysis has 2 endergonic and two exergonic reactions

oEndergonic: glucose to glucose 6-phosphate,

Fructose 6-phosphate tow Fructose 1,2-

bisphosphate

oExergonic: Glyceradehyde 3-phosphate to 1,3-

bisphosphoglycerate (produces NADH), 1,3

bisphosphoglycerate to 3-phosphoglycerate

(really exergonic, produces ATP), 2-

phosphoenolpyruvate to pyruvate (ATP)

Inner membrane of Mitochondria has >70% protein

oIt is impenetrable to basically everything (even

ions and small molecules) except at channels

Outer membrane is ~50% protein

oHas porins which accepts anything small (both

uncharged and ions) through passive transport

into the intermembrane space (same

composition as cytoplasm) through passive

transport

Krebs cycle (Acetyl-S-CoA combines with Oxaloacetate

(C4) to form Citrate (C6)

oOxidation occurs between isocitrate (C6) and a-

Ketogluterate (C5) where an NAD+ is reduced

to NADH and CO2 is released

oOxidation also occurs between a-Ketogluterate

(C5) and Succiny CoA (4C??) where NADH is

made and HS-CoA (conenzyme A) comes into

cycle and CO2 is released

oGTP is made between Succinyl-CoA and

Succinate (C4)

oBetween Succinate (C4) and Fumerate (C4),

FAD is reduced to FADH2

oBetween Malate and Oxaloacetate, NADH is

made

oTotal made (including Pyruvate oxidation is 6

C)2, 8 NADH, 2 FADH2, and 2GTP

In Complex I, 42 polypeptide chain (transmembrane

protein)

Coenzyme Q (lipid soluble since in the middle), 13

polypeptides

Complex III

Then Cytochrome C which carries electron from III to IV

(on intermembrane side)

Complex IV

Complex II (on matrix side) cannot transport H+ since no

transmembrane

pH of matrix is 8, pH of intermembrane space is 7

(cytoplasm is buffer)

All energy from breakdown of glucose (not lost as heat),

but is ultimately used to make ATP

36 ATP made from aerobic respiration

Amino acids enter in cycle depending of R-group, fatty

acids make Acetyl group and glycerol make G3P

When done anaerobically, glycolysis occurs about 10x

faster

Fermentation in yeast goes from pyruvate which is

catalyzed by pyruvate decarboxylase which releases

CO2 and Acetaldehyde (2C or ethanol) which is

catalyzed by alcohol dehydrogenase which also oxidizes

the NADH to NAD+ and creates ethanol as a byproduct

Fermentation in bacteria and human muscle cells is

done by using pyruvate to be catalyzed with lactate

dehydrogenase which oxidizes NADH into NAD+ and

produces lactic acid (3C) which is moved to liver and

converted to glycogen

Electron Transport Chain pauses, not breaks when no

Allosteric regulator can be positive and negative (like

noncompetitive inhibition)

When allosteric regulator is product of later reaction,

called Feedback inhibition

Rate limiting step is the slowest step and in glycolysis, it

is where Fructose 6-phosphate becomes Fructose-1,6-

bisphosphate also known as the first irreversible step

since highly exergonic

Irreversible enzymic reaction that occurs at a branch

point during biosynthesis of some molecules

Rosalind Franklin only said the DNA was helical, width

2nm and sugar and phosphate on outside while bases

on inside

Knocking off nucleotide is hydrolysis, NOT denaturing

Bases are stabilized by H bonds between opposite base

pairs and hydrophobicity causing base stacking

interactions (no enzymes needed to form this structure)

To denature ad heat, acid or base, urea, or formide

Any two nucleic acids despite species or DNA/RNA will

anneal

Melting temperature (when all are halfway undone)

increased with more GC and presence of salt (sharp

melting curve usually 6-8°C

When linear DNA is replicated, a couple base pairs at

the end are not able to be duplicated (this has to do

with primers necessary for DNA replication and okazaki

fragments). Eukaryotic chromosomes therefore have

repeating base pairs at the end called telomeres to

protect any genes from being cut short through

replication

Bacteria have minimal genomes. Circular DNA bypasses

the need for repeating ends on their chromosome(s).

Disadvantage (from a eukaryotic standpoint) is that you

cannot have crossing over with circular DNA.

Circular DNA in mitochondria and chloroplasts!!!

In humans, all of the chromosomes stacked would be 2

meters in length (3 billion base pairs; individual

chromosomes some 250 million)

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MCB Exam 2 Lecture Notes  NAD+ accepts 2e and 1H+ to form NADH (other H+ floats around)  FAD accepts both 2e and 2H+ to form FADH  Bond in NAD+ or NADH connecting the phosphate groups is NOT a phosphodiester bond  In ATP, phosphate groups are unstable meaning that since they are so close to each other, have positive potential energy  Leftover energy in any coupled reaction with ATP is lost as heat  Glycolysis has 2 endergonic and two exergonic reactions o Endergonic: glucose to glucose 6-phosphate, Fructose 6-phosphate tow Fructose 1,2- bisphosphate o Exergonic: Glyceradehyde 3-phosphate to 1,3- bisphosphoglycerate (produces NADH), 1, bisphosphoglycerate to 3-phosphoglycerate (really exergonic, produces ATP), 2- phosphoenolpyruvate to pyruvate (ATP)  Inner membrane of Mitochondria has >70% protein o It is impenetrable to basically everything (even ions and small molecules) except at channels  Outer membrane is ~50% protein o Has porins which accepts anything small (both uncharged and ions) through passive transport into the intermembrane space (same composition as cytoplasm) through passive transport  Krebs cycle (Acetyl-S-CoA combines with Oxaloacetate (C4) to form Citrate (C6) o Oxidation occurs between isocitrate (C6) and a- Ketogluterate (C5) where an NAD+ is reduced to NADH and CO2 is released o Oxidation also occurs between a-Ketogluterate (C5) and Succiny CoA (4C??) where NADH is made and HS-CoA (conenzyme A) comes into cycle and CO2 is released o GTP is made between Succinyl-CoA and Succinate (C4) o Between Succinate (C4) and Fumerate (C4), FAD is reduced to FADH o Between Malate and Oxaloacetate, NADH is made o Total made (including Pyruvate oxidation is 6 C)2, 8 NADH, 2 FADH2, and 2GTP  In Complex I, 42 polypeptide chain (transmembrane protein)  Coenzyme Q (lipid soluble since in the middle), 13 polypeptides  Complex III  Then Cytochrome C which carries electron from III to IV (on intermembrane side)  Complex IV  Complex II (on matrix side) cannot transport H+ since no transmembrane  pH of matrix is 8, pH of intermembrane space is 7 (cytoplasm is buffer)  All energy from breakdown of glucose (not lost as heat), but is ultimately used to make ATP  36 ATP made from aerobic respiration  Amino acids enter in cycle depending of R-group, fatty acids make Acetyl group and glycerol make G3P  When done anaerobically, glycolysis occurs about 10x faster  Fermentation in yeast goes from pyruvate which is catalyzed by pyruvate decarboxylase which releases CO2 and Acetaldehyde (2C or ethanol) which is catalyzed by alcohol dehydrogenase which also oxidizes the NADH to NAD+ and creates ethanol as a byproduct  Fermentation in bacteria and human muscle cells is done by using pyruvate to be catalyzed with lactate dehydrogenase which oxidizes NADH into NAD+ and produces lactic acid (3C) which is moved to liver and converted to glycogen  Electron Transport Chain pauses, not breaks when no O  Allosteric regulator can be positive and negative (like noncompetitive inhibition)  When allosteric regulator is product of later reaction, called Feedback inhibition  Rate limiting step is the slowest step and in glycolysis, it is where Fructose 6-phosphate becomes Fructose-1,6- bisphosphate also known as the first irreversible step since highly exergonic  Irreversible enzymic reaction that occurs at a branch point during biosynthesis of some molecules  Rosalind Franklin only said the DNA was helical, width 2nm and sugar and phosphate on outside while bases on inside  Knocking off nucleotide is hydrolysis, NOT denaturing  Bases are stabilized by H bonds between opposite base pairs and hydrophobicity causing base stacking interactions (no enzymes needed to form this structure)  To denature ad heat, acid or base, urea, or formide  Any two nucleic acids despite species or DNA/RNA will anneal  Melting temperature (when all are halfway undone) increased with more GC and presence of salt (sharp melting curve usually 6-8°C  When linear DNA is replicated, a couple base pairs at the end are not able to be duplicated (this has to do with primers necessary for DNA replication and okazaki fragments). Eukaryotic chromosomes therefore have repeating base pairs at the end called telomeres to protect any genes from being cut short through replication Bacteria have minimal genomes. Circular DNA bypasses the need for repeating ends on their chromosome(s). Disadvantage (from a eukaryotic standpoint) is that you cannot have crossing over with circular DNA.  Circular DNA in mitochondria and chloroplasts!!!  In humans, all of the chromosomes stacked would be 2 meters in length (3 billion base pairs; individual chromosomes some 250 million)

 Topoisomerase relieves supercoiling by breaking phosphodiester backbone and gluing back together (cannot change sequence)  Chromosomes in functional domains of nucleus  Histones about 100 amino acids which are extremely conserved (cows and peas only differ by 2) o + charge o H1 (causes zigzagging), H2A, H2B, H3, H o Rich in a-helix o Histone code – (acetyl, methyl, and phosphate placed by acetylases/deacetylases, methylase/ demethylase, and kinase/phophotases) form random coils o Important for cell to notify cell to unwind or repack  Nucleosomes (146 base pairs wrapped around 1. times; no H1)  Chromatosome (nucleosome including H1 and total of 166 base pairs which gives 10 extra on each side)  Non histone proteins present on 10-nm fibers (different from histone-like proteins in bacteria)  Linker is 20 DNA base pairs long totaling 200 (166+20+20)???  10 nm chromatin fibers “only” shorten DNA by 6x  30nm is either zig zag or phone cord (6 nucleosomes coiled together stacked on top of each other)  Interphase – replicating DNA and growing  M – nuclear division (mitosis); cytokinesis (cell divide)  Euchromatin – MOST IN FORM OF 30 NM FIBERS ; 10% is in more decondensed form for transcription  Heterochromatin – often found at periphery of nucleus  Nucleolus in staining picture is not heterochromatin  30 nm to 300 nm hetrochromatin done by scaffolding proteins  Then to 700 nm which is 1 arm of chromosome which total is 1400 nm  Cohesion holds together sister chromatids???  Telomeric region on all four arms  Proteins bound to end of telomeres to recognize end of linear chromosome  10-30 nm is euchromatin  30+ is heterochromatin  Packed tightly so doesn’t break if pulled apart in M phase  Overall from DNA to chromosome, condensed 50,000 x  DNA binding protein in histones(????)  Bacteria chromosome have a few million base pairs and single ori (no stop signal); 20-50 minutes for replication with 1 ori  If linear chromosome has single ori, takes 3 weeks to get 2 DNA  In DNA replication, new phosphodiester bond created from pyrophosphate being broken off from incoming nucleotide  All nucleic acids (RNA and DNA) can “try” to bond, but polymerase just filters out “bad ones”  Synthesis from only in 5’ to 3’ direction of new strands  DNA polymerases require a free 3’OH group to add  Primase creates primer 3-15 base pairs (technically only need 1 nucleotide to function, but 1 base not stable enough  DNA Polymerase ΙΙΙ (DNA dep DNA synth)  Starting primase RNA haw 3 phosphate attached  DNA pol III continue until run out of fragment or hit an okazaki  Primer about 10 bp, Okazaki about 1000 bp  Reads template 3’ to 5’  DNA pol I – multitasking (kick out RNA [5’ to 3’ exonuclease activity] and put in DNA so slower) (NOTE, these two abilities are of SEPARATE ENZYMATIC ACTIVITIES)  DNA pol III (speed)  Nick = no phosphodiester linkage; gap no nucleotide  Nick provides free 3’ OH group for DNA pol I  After DNA pol 1, nick moved to where end of primase was and is sealed by ligase (which is DEPENDENT ON ATP )  Helicase may cause supercoiling at parts not unwound yet so topoisomerase comes in and fixes it ahead of fork  NOTE: Helicase is DEPENDENT ON ATP  (in picture, it looks like primase hooked up with helicase on lagging strand)  RNA synthesizing enzymes require a free 3’ OH for SYNTHESIS but can hybridize a nucleotide to a nucleic acid strange  1 st^ RNA require energy to be attached which is DEPENDENT ON ATP?  mRNA is used as a way for AMPLIFICATION  RNA polymerase similar to, but not exactly primase  RNA consists of 6 subunits (α 2 ββώσ)) o with σ), called HOLOENZYME o without σ) called CORE ENZYME o doesn’t transcribe entire genome into RNA (difference between replication) o looks for individual genes which start at promoter  -35 region is TTGACA  -10 region is TATAAT  The more a bacteria promoter resembles those regions, the better RNA will bind, which means more RNA and stronger expression  Less similar sequences used as regulation when we only need a little bit of that protein  Terminator is transcribed, not Promoter!  First base is +1, there is no #0 base  Holoenzyme (σ) FACTOR) scans DNA for promoter (- and -10 regions) and when found, noncovalent clamp onto DNA, sigma is released to find other promoter  DNA unwinds as RNA polymerase proceeds and is rewound at the trailing end New RNA chain is displaced (elongation done by Core enzyme)  Coding strand is the non-template strand  Wrong to think that every DNA has one coding and one template, either strand can be used at template depending on location and orientation of promoter

o Stop (UAA, UAG, UGA) then cut (endonuclease activity) after 10-30 bps, then add on AAAAAAAAAA (100-200 A’s) o Nothing depending, RNA synthesizing enzyme o Aids in allowing multiple compies of single protein to be efficiently made from a single eukaryotic mRNA o Provides temporary stability from RNases o Allows circular form so when translation ends, it can start right back again  Trick question warning!! NO BOUND RIBOSOMES IN PROKARYOTES  Genetic Code is Degenerate (each amino acid has a couple combination of nucleotides) but not Ambiguous (each nucleotide codon might encode different amino acids) (although some are not degenerate like Met or Trp  Also (nearly) universal which means different species to use same code combination  Amino acid attachment on tRNA site at 3’ end which contains ACC from top down  tRNAs are about 75-80 bp which folds up intrastrand base pairing with hydrogen bonds  if 5’ tRNA on left and 3’ tRNA (with ACC) on right, then from 5’ to 3’ of mRNA goes from right to left  binding of amino acid to tRNA molecule is VERY specific  requires input of energy provided by HYDROLYSIS OF ATP TO AMP and pyrophosphate which is catalyzed by enzymes called Aminoacyl tRNA synthetase (AARS); this reaction is endergonic  sequence of bases of tRNA means different shapes allowing AARS to recognize the tRNA it’s looking for (it is larger so it can feel both anti codon and attachment site)  AARS ½ add amino acid to 2’ and the other to 3’  2’ site of carbon when adenine is flipped upside down is on right side  When this bond is made between adenine and amino acid, later broken to allow attach to next amino acid  Ribosomes in prokaryotes and eukaryotes are functionally identical but structurally different o When put together, they take up less space o Prokaryotic 70S ribosome  50S  23 s and 5 s (120 bp) rRNA’s  34 proteins  30S  16 s rRNA (1500 bp)  21 proteins o Eukaryotic 80S ribosome  60S  28 s, 5.8 s, and 5 s rRNA  ~45 proteins  40 s  18 s rRNA  ~30 proteins  The 3’ end of 16 s rRNA (part of small ribosomal subunit of prokaryotes) is single stranded and not intracellularly bound o 3’ end of 16 S rRNA locks with 5’ end of mRNA just downstream ~10bp there is an AUG there  Instrastrand base paring present in mRNA, tRNA, and rRNA  Archae have Met NOT fMET  +fMET-tRNAfMET  (actually attached) (supposed to be attached)  Start of mRNA--Shine Dalgarno----~10nt----AUG---mRNA  Shine Dalgarno sequence conserved in EVERY BACTERIA  Polycistronic – all PROKARYOTES, not just bacteria carry information for multiple protein products  Start with untranslated region, SD protein, stop, UTR, SD, protein… etc.  Different regions mRNA can be translated by same or different protein  These proteins are completely different from other sequences  In Eukaryotes, small ribosomal subunit (40 s) binds to cap and finds nearest AUG and is this Monocistronic  Initiation of Translation requires mRNA, small subunit, charged tRNA, INITATION FACTORS, and GTP !! o The Initiation factors and GTP recruit large subunit; after done, they are both released  Elongation phase requires EF-TU which helps get tRNA to ribosome o Any tRNA can try to fit in A site o Slight conformation change when paired “rocking mechanism” o GTP provides “quality control” by helping next amino acid is right; GTP is pushed into crevice of ribosome into active site of GTPase in which the energy is used to make elongation factor let go only when appropriate pairing is made o Peptidyl transferase is enzyme which breaks bond of amino acid in P site forms new bond between polypeptide and new amino acid (Ribozyme activity)  Translocation o Requires another EF (EF-G) and GTP in order to move ribosome along mRNA o Ribosome moves toward 3 bases toward the 3’ end of the mRNA displacing the uncharged tRNA o N-terminus is not connected to amino acid, but simply hanging  Termination involves release factors (not hybridization where protein interacts w/ nucleic acid)  Polysome possible where more than 1 ribosome working 1 mRNA at the same time  “cotranscriptional translation” only possible in bacteria (prokaryotes)

Exam 2 Study Guide - Molecular and Cellular Basis of Life | MCB 150, Study notes of Biology

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 Multiple RNA polymerases can transcribe at once