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Watson and Crick accomplishment
structure of DNA
- "Transforming Principle".
- mixed heat-killed S (virulent) with live R (non-virulent) and got dead mice.
- Saw encapsulated (S) vs non-encapsulated (R) Strep pneumoniae.
- Capsule = polysaccharide = virulent
- unencapsulated (R) is not virulent.
"S" form Strep pneumoniae
"smooth", encapsulated, polysaccharide coat, VIRULENT
"R" form Strep pneumoniae
"Rough" unencapsulated, non-virulent
Avery, MacLeod and McCarty
- DNA is the transforming principle
- Took Griffiths' S and R experiment and split all the heat-killed S parts, discovered it was DNA (removed DNA and mouse lived).
- Phage genetic material is DNA
- Made sulfer and phosphorus radioactive at dif times. Sulfer (amino acids) stayed in phage shell, phosphorus (DNA) was injected into bacteria.
- Chase is MARTHA, female
3 properties of hereditary materials
- faithful replication (high fidelity)
- have info
- can change but generally remain stable
building blocks of DNA
- deoxyribose (sugar)
- one of four nitrogenous bases
- A, G (double rings)
- A has NH2
- G has =O, H, NH2
- C, G (single rings)
- C has NH2, =O
- T has CH2, =O, H, =O
- double ring with NH2 hanging off
- pairs to T (or U) with 2 bonds (loosest)
- purine, double ring with =O, H and NH2 hanging off
- Pairs to C, 3 bonds (tightest)
- Pyrimidine, single ring with NH2, =O hanging off.
- Pairs to G with three bonds, tightest
- pyramidine, single ring with CH2, =O, H, =O hanging off
- pairs to A with 2 bonds
- DNA only
draw a nucleotide
phosphate circle - furanose ring (one OH opposite O) - nitrogenous base
a nucleotide that is missing the phosphate
found rule that total amount of pyramidine always equals the total amount of purine (therefore A-T, C-G)
photograph 51, took photo that showed Watson and Crick about the helix. Used x-ray diffraction to show that bases are on the INSIDE.
structure of DNA
- backbone of sugar-phosphate held together by phosphodiester linkages.
- Side-by-side chains of nucleotides held together by H-bonds.
bond for backbone of DNA
phosphodiester (covalent) bond holds phosphate-and-sugar backbone together.
bond for nucleotides in DNA
hydrogen bonds, purine bonds to pyramidine
Phosphate connects to ____' end
3' C (has OH)
phosphodiester bonds connect to what part?
5' or 3'
for strength in DNA want ____
for dynamic DNA want
- where proteins sit down on DNA
- big cross in helix when you look straight at it
helix of single strand
none. The shape depends on the pairing/stacking of the base pairs
semiconservative model of replication
one original strand goes to each daughter molecule
conservative model of replication
one molecule stays complete, 2nd molecule made from completely new material
dispersive model of replication
chunks of old molecule in both new molecules
- DNA is copied by semiconservative replication
- use radioactive nitrogen to figure out if DNA copies by conservative, semiconservative or dispersive model (blue and yellow). Centrifuged, radioactive is light.
discovered DNA polymerase I. Reads DNA strand it's on (3'-5'), makes new strand (5'-3'), proofreads.
- Reads, makes new strands and proofreads.
- Reads from 3'-5', makes from 5'-3' (ANTIPARALLEL)
- discovered by Arthur Kornberg (pol 1)
- Links new base to 5'
- There are 5 DNA polymerases in E.coli
Necessary DNA polymerase
a DNA polymerase that cuts nucleic acid from the ends (as proofreading)
a DNA polymerase that cuts nucleic acid from the center (for proofreading)
where DNA unwinds to be replicated. Enzymes STAY IN FORK
synthesizes DNA (5'-3') toward replication fork, easy, smooth.
synthesizes DNA (5'-3') away from fork, has to keep going back (Okazaki fragments)
- How lagging strand synthesizes DNA.
- 1. primase makes RNA primer
- 2. polymerase 3 makes DNA
- 3. Polymerase 1 (exonuclease), cuts up RNA primers
- 4. ligase comes in and seals ends together
- very few mistakes. DNA has less than 1 error per 10^10 nucleotides (polymerases 1 and 3 are proofreaders)
- RNA makes many more errors, okay because of short half-life
exonuclease activity removes wrong base to give another chance. Still wrong sometimes.
- the proteins working at the replication fork
- helicase, topoisomerase, primase, DNA polymerase, beta clamp, single-stranded binding proteins
separates two strands of DNA at replication fork
works above replication fork in DNA synthesis, cuts one strand, lets it unravel, puts it back together to prevent supercoiling (endonuclease activity, relieves pressure)
puts down RNA primer in DNA synthesis at replication fork
how long enzymes work, usually fall off pretty quickly. DNA polymerase III needs beta clamp to keep it on in DNA synthesis at replication fork
helps DNA polymerase III stay on DNA strand in DNA synthesis at replication fork
single-stranded binding proteins (SSBP)
white pearls, protect single-stranded DNA until new strand is formed. Mostly important on lagging strand because leading is so fast.
- one of the topoisomerases
- removes extra twists in DNA to prevent supercoiling during DNA synthesis
- origin of replication in bacterial chromosomes.
- Called OriC in e coli (origin of chromosomes)
- Has dnaA boxes behind A-T-rich region
- helicases move in both directions to speed things up, replisome replicates DNA
- Origin of chromosome, in ecoli.
- Like OriR (replication) in other bacterial chromosomes
- dnaA boxes behind an A-T-rich area, helicases go in two directions to speed things up, replisome replicates DNA.
Eukaryotic vs prokaryotic replisome
- ecoli has 13 componenets
- yeast and mammals have 27 components, which must be disassembled from histones and reassembled in daughter molecules.
- yeast has 400 oris, humans have thousands of oris
Can DNA come from RNA (arrow backwards)?
RNA to DNA by reverse trascriptase, only found in retroviruses so far
- process of synthesis of RNA (transcript) from DNA
- First step in transfer of info from genes to products
- intermediate molecule that is a copy of a gene using DNA sequence as a guide
- single stranded, more flexible than DNA
- INTRAMOLECULAR BASE PAIRING (complex shapes)
- Ribose sugar, extra OH important.
not true for eukaryotes. 1 gene can make 64,000 proteins due to splicing. 20,000 genes = 100,000 proteins in humans
- When making genes, not all exons have to stay in. Different combinations make different proteins
- responsible for differentiation of cells
- How complex organisms are different than prokaryotes
STAY IN, get made into proteins
GET CUT OUT, garbage
different results you get with alternative splicing. Helps in differentiation of cells
How do proteins know where to sit down on dna?
bind in major groove to specific sequences/base pairs.
RNA polymerase activity
sits down on DNA, makes a tiny separation (not like replication fork) and reads one side, makes complimentary side
How does single-stranded RNA stay stable?
- INTRAmolecular base pairing, folds into complicated shapes
- 5'cap and 3' poly-A tail (like a coat, temporary protection)
- pyrimidine, one right with =O, N, =O haniging off
- binds to A with 2 bonds. Can bind to G in intramolecular, weakest bond.
- RNA only
RNA can catalyze reactions (ribozymes)
- messenger RNA
- serve as intermediary that passes info from DNA to protein.
- RNA IS the final product, no protein made.
- transfer info, process other RNAs, regulate RNA and protein levels
- tRNA, RNAi
- transfer RNA
- bring amino acid to mRNA during translation
- ribosomal RNA
- make up ribosomes.
- HIGHEST PERCENT OF RNA is rRNA
- small nuclear RNAs
- part of processing system of RNAs (eukaryotes only)
- can combine with proteins to form spliceosome that removes introns from eukaryotic mRNA
- regulate amount of protein produced by genes
- functional RNA
- small interfering RNA
- protect the integrity of genomes. Inhibit the production of viruses. Prevent the spread of transposons (plants)
- piwi-interacting RNAs
- protect integrity of genome. Prevent spread of transposons
lncRNA or ncRNA
- long noncoding RNAs
- we don't know what they do
- made all the time in high amounts.
- tRNA, rRNA, snRNA
RNA made as needed
miRNA, siRNA, piRNA, lncRNA
Elliot Volkin and Lawrence Astrachan
- radioactive "pulse-chase" experiment to prove that RNA is intermediate between DNA and protein
- phages use RNA to copy in ecoli
- RNA short-lived
How RNA polymerases increase variance
- 1. Genes are on one strand of DNA. RNA only reads one strand (read 3'-5', make 5'-3')
- 2. alternative splicing
higher fidelity in DNA or RNA?
DNA, RNA screwed up all the time, short lifespan and lots of them so we don't care much
nontemplate vs template strand in RNA synthesis
RNA reads the template strand, makes a line that is like the nontemplate or coding strand.
3 stages of transcription
- initiation-RNA polymerase sits down at promoter.
- termination-hits transcription termination (we don't know in eukaryotes).
specific promoter sequences in e coli
- -35, -10 ("tata box")
- proteins recognize a promoter by a specific sequence. well-conserved, must have both to get started.
- first protein to sit down in RNA transcription in BACTERIA. Does not move with RNA polymerase
- shaped like a crescent, two points bind to -35 and -10
- Sits down, calls RNA polymerase, only initiates when it should. Helps spread DNA to start transcription. Release enzyme to let RNAP leave promoter. Leaves or waits for next
- untranslated region
- space between promoter and gene. Part of mRNA but not part of protein. Where sigma factor sits down.
6 subunits of transcription initiation in prokaryotes
- heterodimer: sigma factor (comes and goes), RNA polymerase with 2 alpha, 2 beta, one omega.
5 subunits of RNA polymerase heterodimer (RNAP)
- 2 alpha, assemble enzyme (regulatory proteins)
- beta, catalytic region
- beta prime - binds DNA
- omega - assembly
- "crab claw", RNAP, DNA fed through center of claw at "active center cleft"
starter codon in most proteins
untranslated region. Region between promoter and gene, where ribosome sits down. Part of the mRNA but not the protein.
the -35 and -10 regions at the beginning of the transcription of a gene
Bacterial RNA polymerase
does it all. Unwinds, etc. works entirely by itself in transcription bubble.
Bacterial RNA polymerase termination
- Intrinsic or Rho-dependant.
- Intrinsic: hairpin loop, stem-loop, followed by UUU sequence
- Rho-dependant: rho protein climbs up mRNA and ends, not sure how
- intrinsic RNA polymerase termination of transcription
- Intramolecular binding between intrinsic terminators causes stem-loop/hairpin turn followed by long stretch of Us (so break apart more easily). Unstable, falls off to stop transcribing.
- Termination is in the structure, doesn't need anything else.
Rho-dependent transcriptional termination
- no need for stem-loop or hairpin turns. Rho protein binds to RUT site (rho utilization terminator, where Rho binds)
- binds, moves up mRNA until it gets into transcription bubble, dissociates everything, breaks apart, ends transcription.
- Not sure how, 3 hypotheses (pushes RNAP off, pulls RNA out of RNAP or makes conformational change)
Why is transcription more complicated in eukaryotes?
- more genes and more non-coding DNA
- nucleus requres RNA processing
- Histone proteins can block
"coupling" and "uncoupled" and examples
- Happening at the same time.
- in prokaryotes mRNA is already being translated as it finishes transcribing--coupled
- in eukaryotes, transcription is in nucleus and ribsomes are outside, don't happen at same time. Uncoupled.
eukaryotic RNAP I encodes for
eukaryotic RNAP II codes for
all protein-encoding genes (mRNA)
eukaryotic RNAP III codes for
small functional RNA genes (RNAi, microRNA)
- general transcription factors
- same function as sigma factor. Recognize and bind to sequences in the promotor (tata box or to other GTFs, attract RNAP
- TFIIA, B, etc (for RNAPII)
eukaryotic preinitiation complex (PIC)
6 GTFs (general transcription factors) and RNAP II.
tata binding protein, in eukaryotic transcription. First to bind (sigma-factor-like, transcription factor) with TFIID. Recruit more
Compare and contrast between prokaryotic and eukaryotic transcription.
Carboxy tail domain
long string of phosphates at bottom. Central to processing (5' cap, poly-A 3' tail, splicing out introns)
- guanosine witha methyl on it.
- 2 functions: protects from degredation in cytosol and recognized by ribosome as the place to start
- put on by carboxy tail domain
- After termination at AAUAAA or AUUAAA, RNA cut and 150-200 adenines added (poly(A) tail)
- Protects and ends.
- removal of introns and joining of exons
- RNA splicing performed by spliceosome
- alternative splicing causes 20,000 genes making 100,000 proteins. 2% of genome encodes proteins. Rest is noncodingRNA
- Splicing. Start of intron to be removed (5') is always GU and end (3') is always AG. Branch point MUST be an A.
- Recognized by 5 small snRNP
snRNP (small nuclear ribonucleoproteins) and 100 other proteins, hugely complex.
- small nuclear ribonucleoproteins
- functional RNA (U1, U2, U4, U5, U6) and protein
- recognize introns that need to be removed (GU-AG rule)
When some exons are kept and others are not, adds variety to the proteins that one gene can code for. Many different spliceoforms. How higher organisms get their complexity
the synthesis of a polypeptide directed by the RNA sequence
- functional RNA
- components of translation machinery
- translate three-nucleotide codon into amino acid
- brings amino acid to ribosome
- There are 20 tRNA types, one for each AA
- with rRNA, makes up 95% of RNA
- fuctional RNA
- major component of ribosomes (with proteins and other RNA)
- with tRNA, makes up 95% of RNA
- main determinants of biological form and function
- influence shape, color, size, behavior, physiology of organisms
- composed of amino acid monomers
- chain is a polypeptide
- have an amino end and a carboxy end
holds amino acids together, made by dehydration synthesis
levels of protein structure/organization
- primary: sequence of AAs
- secondary: a helix or beta pleated sheet
- tertiary: conformation
- quaternary: subunit structure (homodimer, heterodimer)
overlapping genetic code
original hypothesis that codons overlapped and each nucleotide was used in multiple codons. WRONG
non-overlapping genetic code
correct hypothesis that each codon is separate, each nucleotide used only once. Each triplet codon codes for an AA
Sydney Brenner and Francis Crick
used proflavin to delete single nucleotides to study effect of mutation on T4 phage. Discovered frameshift mutation
when a nucleotide is added or subtracted. Throws entire code off by one letter, rest of the protein becomes nonfunctional. Sticking something in same area should restore frame
synthesized mRNA in just a string of Us, added in ribosomes and got a bunch of Phe. Learned the code for Phe
structure of tRNA
cloverleaf. Three stem-loops and one stem (3' hydroxyl group), where AA attaches. bottom is anticodon loop, which has the complimentary strand for the codon for the AA it is carrying.
bottom of tRNA molecule, which has complimentary codon (but 3'-5') for AA codon that tRNA is carrying.
- attaches amino acid to it's tRNA.
- AA pocket extremely specific. tRNA binding site slightly less due to redundancy of of genetic code
- One aminoacyl-tRNA synthase for each amino acid/tRNA (20)
allows tRNA to recognize 2 codons. Sometimes tRNA can bring AA to different codons which don't quite match.
where codon and anticodon meet in ribosome. Must match
peptidyl transferase center
where peptide bond is being made in ribosome, must match.
- bacterial mRNA sequence that calls part of ribosome, tells it where to start.
- like Kozak sequence in eukaryotes. Surrounds AUG (met, starter codon) to show ribosome.
eukaryotic mRNA sequence that calls part of ribosome, tells it where to start. like Shine-dalgarno sequence in bacteria. Surrounds AUG (met, starter codon) to show ribosome.
in ribosome, where amino acids are turning into growing peptide chain
brings together tRNA and mRNA to translate nucleotide sequence of an mRNA into the AA sequence of a protein
3 major sites on a ribosome
- A, P, E
- A = aminoacyl
- P = peptidal
- E = exit
- tRNA comes into A site, leaves when hits the E site. Peptidal site is where AAs are joined.
termination of translation
stop codon brings in release factor