Genetics Wells exam 3

Translation initiation PROKARYOTE

"30s complex" is made of initiation factors (IF1 + IF2 + IF3) + GTP + 30s unit. The 30s complex hooks onto the mRNA strand (the shino-delgrano sequence lines it up to attach) and the tRNA attached to f-met goes on top. The 50s unit attaches on top of the whole deal. E, P, A sites are kinda holes.

Garrod 1909

Described how genes control the biochemistry of the cell, leading to phenotypes (and sometimes "inborn errors of metabolism").

Mismatch repair

Fixes: tautomer-cased problems, micro-deletions, duplications Endonuclease nicks one strand (~1000 BP away), exonuclease cuts out segment, DNA polymerase adds on to 3' end, DNA ligase joins the strands.

Stem cells

Gamete DNA doesn't get shorter (with every generation) because telomerase expands the 3' end (using the complement to TTAGGG) and loops it around to attach it to the 5' end, then splices it off so that the telomere is expanded on both strands.

Bateson 1902

Gene control of biochemistry: he was the first to postulate that alcaptonurea (recessive autosomal, black pee, black cartilage, prone to arthritis) was caused by a gene.

Purine keto:

Guanine (G)

Sumner 1926

Isolated the first pure crystalline protein (urase), did x-ray crystallography to view secondary structure.

Tryptophan operon (prokaryotes)

Negative repressible (when tryptophan present, gene turned off because tryptophan makes the regulatory protein bind to the O site). -structural genes E and D make 3 proteins -structural genes B and A make tryptophan synthetase -after the O site, there's an attenuator region. If a lot of tryptophan present in the system, mRNA makes a CG hairpin loop followed by UUU (for rho-independent termination), and the RNA stops transcribing. Turns gene off.

Pasteur/Büchner weird stuff

Pasteur: cell --> EtOH Büchner: parts of cells -->EtOH

Arabinose operon (prokaryotes)

Positive inducible by arabinose. -when arabinose is bound to the regulatory protein (a C-C dimer), the dimer opens up a little and can bind to the O site, turning on BAD structural genes. ALSO, negative inducible by arabinose--can stop the O2 (second operator site) from loop around and bind to the first initiator site, blocking the promoter site. Either way, arabinose turns the gene on when present.

Eukaryote transcription INITIATION

RNA polymerase II has transcription factors: TFII-A, TFII-B, TFII-C, TFII-D, and TFII-E. (D recognizes the promoter region). In promoter region, TATA box (-30), CAT box (-80), GC repeats (-110). Downstream from zero, in the gene, there is an untranslated region followed by exon/intron --> another untranslated region.

Non-conservative DNA replication:

Random parts of parent strands acts as templates for random parts of new strands.

Avery, McLoud, McCarty 1944

Re-did Griffith's 1928 experiment but on a petri dish instead of a mouse. Saw that the heat-killed smooth-coat bacteria broke up into fragments (coat, protein, DNA, RNA). Only the DNA part, combined with the live rough-coat bacteria, resulted in live smooth-coat bacteria.

Translation termination

Release factors release peptide (amino acid) from tRNA when they hit a stop codon

Sticky end problem

The ends of telomeres have a few unmatched base pairs where the DNA primer was sitting before it moved, and those ends will attach to ANYTHING. Telomere loops protect sticky ends by curling around on each other. T loop is inside (3' end actually attaches), D loop is outside (along for the ride)

Enhancer/silencer region eukaryotes

There's an enhancer region a ways upstream of the promoter region, so transcription factors loop up the DNA in the middle to bring the regions closer together and make transcription happen FASTER :) Silencers block the enhancer region to slow down/stop the transcription.

Holliday structures

Two homologous chromosomes have endonuclease nicks in them, and "strand displacement" occurs as they hook together in a misguided attempt to repair the nicks. Then the chromosomes are hooked together in an X-like holliday structure; they can either be cut so that the original chromosome is restored, or cut so that a crossover event occurs.

Stop codons

UAA, UAG, UGA

Primary structure of protein

amino acid sequence

Cairns 1962

autoradiograph of bacterial DNA--since bacterial DNA is circular, there is one origin and DNA unwinds/replicates in both directions (always adding on to the 3' end).

Prokaryote transcription ELONGATION

building

UV light

causes thymine dimers--adjacent thymines on a DNA strand will bind with EACH OTHER, instead of their A's, and distorts the strand.

Conservative DNA replication:

double parent strands create an identical double strand

Wobble theory

explains why the third base is the most variable when two codons code for the same amino acid. tRNA can reach first two bases really well, but third base not so much--it can't reach very well, so it's the most likely to be different but still make the same amino acid.

Translation elongation

fmet-tRNA is sitting in the P site of the EPA sites. An amino acid comes along from the right to be translated, and the fmat hops over onto it. The whole system shifts to the left, so now the tRNA is on the E site, with two amino acids on the P and A sites. The fmet hops over to the right another time, and the system shifts to the left again, and it keeps hopping, creating a chain. Movement of ribosome along mRNA is funded by GTP.

Tertiary structure of protein

folding patterns (especially pockets)

Prokaryote gene regulation

polycistronic genome--many genes produce peptides (biochem pathway). Regulation systems: -negative= R protein binding to O site turns gene OFF -positive= R protein binding to O site turns gene ON -inducible= presence of a chemical binds to R protein and does something to turn gene ON when chemical present -repressible= presence of a chemical binds to R protein and does something to turn gene OFF when chemical present

Frame-shift mutaiton

single-base addition or deletion (such as in acridine dyes experiment)

transition

switching a purine for a purine, or vice-versa

transversion

switching a pyrimadine for a purine, or vice versa

RNA polymerase II

transcribes mRNA and snRNA (eukaryotes)

RNA polymerase I

transcribes rRNA (eukaryotes)

RNA polymerase III

transcribes tRNA and 5s small rRNA

Tautomer matching

when a base switches to its tautomer, it bonds with the normal form of the WRONG base (like A-imino with C-keto)

Translation setup

-in eukaryotes, the 7-methyl guanine 5' cap is there to protect the mRNA on its way to the cytoplasm. -rRNA goes to the ribosome. Big initial gets cut into 23s + 5s (goes to 50s unit ribosome) and 16s (goes to 30s unit ribosome). -tRNA 40% base cut out, base modification (has to do with guanine methylation). Guanine modified into hypoxanthine (nucleoside of hypoxanthine is inosine). Uracil modified into pseudouracil (different sugar placement) and thiouracil. -tRNA folds, has anticodon on the bottom. Amino acid + ATP join to tRNA, --> tRNA-aa and AMP.

Translation initiation EUKARYOTE

1. 40s unit attaches to eukaryote initiation factors (EIF1 and EIF3) 2. EIF2 + GTP + tRNA-fmet together form the tRNA-fmet complex 3. A 40s-tRNA-fmet complex forms 4. EIF4 attaches the mRNA to that complex (the EIF4 recognizes 5'cap) 5. The complex walks along the mRNA until it hits the AUG codon, using ATP as energy 6. EIF5 links the 60s unit to the 40s after it hits the AUG.

DNA polymerase 3 (DNA poly 3)

A dimer that has 2 sides to replicate DNA so that it can do two strands at the same time. Since DNA strands are antipolar, one side slides through and replicates just fine, but other side ("lagging strand") has to loop around & be made discontinuously with okasaki fragments.

Secondary structure of protein

Alpha helices and Beta waves

Bubble model of DNA replication

At specific sequences (origin sites), gyrases and helicases open up the DNA strands and a bunch of proteins hold them apart while RNA polymerase builds from the RNA primer, then DNA polymerase 3 builds complementary DNA onto the RNA strand. RNA polymerase eventually puts down a new RNA primer; when the new DNA runs into it, the DNA polymerase 3 hops off and DNA polymerase 1 takes over (chopping away the RNA primer as it goes and replacing it with new DNA). When it finally hit new DNA, it hops off and DNA ligase joins the two fragments.

Hayflick limit

Cells divide about 50 times and then they die, because a little bit of the telomere (TTAGGG repeats) is lost as the attachment side of DNA primer with each cell division. When you start losing vital DNA (not junk sequences), the cell is destroyed.

4 bases, 20 amino acids

Codons (words) are formed by groups of 3 bases to form 64 possible combinations or words. This is the smallest number of bases per codon needed to make a non-ambiguous code.

Sub and Horowitz 1944

Created three mutants of neurospora. Arginine biochemical pathway goes: precursos->oranthine->citrilline->arginine. Mutant I: could only grow if added Arginine to media, because mutation was in the last step of the pathway. Mutant II: could grow with citrilline OR arginine (second step) Mutant III: Could grow with oranthine, citrilline, arginine (first step). This shows: one gene per enzyme, because three mutation appeared in the same pathway.

Griffith 1928

Diplococcus pneumonia experiment. Took smooth-coat (killed off mice) and rough-coat (defective, not dangerous to mouse) strains II and III. Found that heat-killed smooth mixed with alive rough-coat would KILL THE MICE--the smooth-coat did the job through "transformation," where genes were taken up by the other bacteria and expressed in genome.

Universal code

Every organism (prokaryote, eukaryote) uses the same codons-- except mitochondria, chloroplasts, protozoa. Typically when two codons code for the same AA, it's the third base that can vary.

Alkylating agents

Example: Chlorine gas (mustard gas). Attacks keto groups, throws a methyl or ethyl on it. Such as ethyl methyl sulfonate: it adds methyls like crazy (lab example)

Hershey and Chase 1952

Experimented by making s35 (radioactive protein) and p35 (radioactive DNA) and creating viruses and mixing them with bacteria. Put the bacteria in a blender to knock off the proteins, centrifuge: the pellet (protein) isn't radioactive, but the supernatant (DNA) is radioactive, showing that p35 is the only thing that makes it radioactive (DNA is genetic substance).

Crick

Experimented with T4 virus, added acidine dyes that sit between bases and act as single-base additions. -2, -1, +1, and +2 were all seriously mutated in the DNA, but -3 and +3 were essentially back to normal, proving that the reading frame is 3 codons (no commas or spaces)

Zinder and Lederburg 1952

Experimented with salmonella. Strain I = met+, thr- (can make its own methionine, needs to be fed threonine), while strain II = met-, thr+. If you combine Strains I & II on a neutral media, you get some met+ thr+ growing! Because of conjugation, transformation? No, repeated Griffith's heat-killed experiment and it only worked with heat-killed strain I. It happened because of "TRANSDUCTION"--a virus associated with strain I that when heat-killed released a ton of bacterial genes, carried the host genes to other strain.

Messelsohn and Stahl 1958

Experimented with semiconservative/ nonconservative/ conservative DNA replication. Created "heavy DNA" made with N15 isotope, and "light DNA" made with N14 normal nitrogen. Let one round of DNA replication occur and measured in a density gradient: saw only 1 band of DNA (all same density, so we know it's not conservative). Let DNA replicate a second round, saw two bands of DNA at two densities--proved that replication is semiconservative.

Nirenberg

First to make mRNA in vitro to experiment. UUU-phenylalanine AAA-lysine CCC-proline GGG-NOTHING, bond to each other He combined 1 A : 5 C, calculated ratios of expected codons, found ratios of created amino acids, and matched of percentages to de-code a bunch of codons.

Nucleotide excision repair

Fixes: T-T dimers (*bacteria have "bacteria photolyase" which breaks the bond, but mammals don't) XP proteins (a suite of about 10) clip on either side of the dimer (~30 BPs in either direction), exonuclease cuts out, polymerase rewrites, ligase joins

Base Excision Repair

Fixes: deaminations, oxidations (8-oxo G), alkylations, AP sites. Glycosylase family recognizes the type of error and strips the base off the sugar, turning every error into an AP site. Endonuclease makes either: -short patch (nick next to AP site, DNA polymerase puts in a new nucleotide, ligase connects) -long patch (nick is farther away 20-50 bases, DNA polymerase comes in from cut point and starts to replace. Flap endonuclease, FEN, cuts off the flap, ligase joins).

Okasaki

Found that eukaryotic chromosomes have multiple origins and replication is also bi-directional. ZIPPER MODEL: in one direction, replication is continuous; in the other direction it's made in pieces, always 5'-3' as it unravels, finding new primers

Base analogs

Just a base with something else thrown on it (like 5-bromo Uracil, 2-amino purine, 5-fluoro Uracil)

Lactose operon (prokaryotes)

Negative inducible by lactose AND positive inducible by glucose. -regulatory protein = I -structural genes = Y (lactose permease, brings lactose into cell), Z (B galactosidase, chops lactose into galactose and glucose), and A (B galactoside transacetylase, puts acetyl group on galactose, not super essential in breakdown path). -When glucose is NOT present, AMP --> cAMP, binds to CAP protein. The CAP protein is able to bind to the CAP O site, turning the gene on. Therefore, when glucose IS present, AMP-->ATP, and the CAP gene is unable to bind to the CAP O site. -no glucose, yes lactose: gene ON. -all other combos: gene OFF.

Beetle and Tatum 1941

Neurospora (vitamin B - or + reated with x-ray mutation) proved Mendelian theory of inheritance -- the gold standard of genetics

Deamination

Nitrous acid (HNO2) can take away the amino and switch it with a keto. Example: A--> hypoxanthine C --> uracil G --> xanthine

Acetobularia

Single cell algae (quite large). Used in experiment: chop off nucleus, feed radioactive stuff to the cytoplasm, and get radioactive protein synthesized. Therefore, protein synthesis happens in the cytoplasm (RNA is an intermediate that goes out of the nucleus and does synthesis)

Taylor 1960

Tested to see if DNA replication was the same in eukaryotes as bacteria. Used a thin film of photoemulsion so that when developed, film will be clear except where exposed to radiation. Used plants: p32 = radioactive DNA, mixed with p31 (non-radioactive DNA), after one cell division you see that BOTH chromatids appear radioactive on plate (meaning one strand of each is radioactive). Proves that eukaryotes are semi-conservative.

1945 genetic substance

Thought: genes are made of proteins, because DNA is too small and simple (even though experiments say otherwise).

5FU

Usually, it's easy to make uracil (for RNA) but difficult to make thymine (for DNA). Usually: U--> UMP (via thymidine kinase) --> UDP --> UTP --> RNA. Instead of having a whole big process for T, you can go: U-->UMP--> UDP --> RNR --> dUDP --> dUTP--> dUMP (via dUTPase) --> TMP (via TS) --> TDP --> TTP --> DNA and make thymine out of uracil's process. If you add 5FU to the system it will bind irreversibly to TS and stop the system from making thymine. In cancer patients, it's a good way to starve the new DNA of Thymine and stop replication.

U-tube experiment

Zinder and Ledenburg further proved the model of bacterial transduction by having strain I & strain II on opposite sides of a U-tube with a bacterial filter in between (and DNAase to chew up any DEAD DNA, not in a cell) -- on the Strain II side, met+ thr+ appeared, showing that live DNA (in the form of a virus) was able to get through the filter and transform the other bacteria!

Prokaryote transcription INITIATION

-2 critical regions in order for RNA polymerase to recognize the promoter regions: -10 TATAAT, -35 TTGACA. -RNA polymerase = aaBB'w. BB' does transcription. aa does assembly and recognition of the promoter region. w does stability. -Sigma factor binds to RNA polymerase and helps it recognize the promoter region, but it binds too much--that's why you have ~10 false starts because it's like an anchor.

eukaryote gene regulation

-Acetylation of histone proteins in nucleosomes activates genes (turn on) because the histone proteins don't associate as strongly -Methylization of cytosine in CPG islands (just poly CG areas next to promoter regions) turns genes off (more methyls = stronger turn-off by preventing transcription factors from finding the promoter region)

Kornberg 1957 (Nobel Prize)

-Built DNA in vitro, trying to see whether DNA is added to the 5' end or the 3' end. Built DNA using only A, T, and G. When he got to a place where a C was needed, he added C tagged with p32 (radioactive). Then added an enzyme to cut the 5'-P bond and found that new DNA is added on the the 3' end. -Found that DNA molecules are antipolar using the same method--the radioactive P wasn't shifted one nucleotide up.

Myc/Max system eukaryotes

-Myc can't bind to DNA, CAN turn on transcription (and can't form homo dimer) -Max CAN bind to DNA, but can't activate transcription. 2 dimers possible: 1. Myc-Max: binds to DNA and activates the gene 2. Max-max: binds to DNA and turns gene OFF Therefore, if the cell only makes Mac, it turns the gene off.

Eukaryote transcription ELONGATION

-Remove introns (junk genes). -Aborted starts: system won't proceed without the 5' cap (mGppp, or methylated guanine triphosphate)

Tautomers

-amino form switched to imino form (NH==C--NH, instead of N==C--NH2) keto form switched to enol form (carbonyl group switched to -OH)

Watson and Crick model of DNA:

-double helix (two linked strands) -bases point inward to hold it together (sugar-phosphate backbone is OUTSIDE) -antipolar arrangement (3' linked to 5') so that bases can pair AT, GC

Eukaryote chromatin structure

-euchromatin=uncoiled, genes turned ON -heterochromatin=very coiled, genes turned OFF. -DNA wound around 8 histone proteins--nucleosomes--make strands shorter and stronger but must unpack before transcription can occur.

negative repressible

A chemical binds to the R protein, allowing it to bind to the O site. Gene turns OFF.

positive inducible

A chemical binds to the R protein, allowing it to bind to the O site. Gene turns ON

positive repressible

A chemical binds to the R protein, preventing it from binding to the O site. Gene turns OFF.

negative inducible

A chemical binds to the R protein, preventing it from binding to the O site. Gene turns ON

Intron splicing

A spliceosome, or snRNP, travels along and lops off any looped-up introns, leaving the exons along.

Start codon

AUG

Purine amino:

Adenine (A)

Eukaryote transcription TERMINATION

After an untranslated region, a 200aa tail is put on the 3' end. If tail is absent, entire strand get cut up. AAGAAA cut site right before the 3' cap.

3 DNA repair mechanisms (single-strand problems)

BER, MMR, NER

Guanine --> 8-oxo Guanine

Caused by x-rays and oxygen radicals. Gets an extra carbonyl group at position 8, causes a transversion by pairing up with A instead of C.

Nirenberg continued

Created mRNA using only two bases to figure out more codons.

Pyrimadine amino:

Cytosine (C)

Semi-conservative DNA replication:

DNA helix splits, replicates, each replicated half is half of a new molecule

Prokaryote transcription TERMINATION

1. Rho-dependent: rho (p) peptide binds to a sequence of the transcribed RNA, walks along until it hits the RNA polymerase and breaks off the RNA polymerase. 2. Rho-independent: GC-rich region of transcribed RNA, flips around and binds in hairpin loop followed by U's. RNA polymerase falls off.

Pyrimadine keto:

Thymine (T) -has a methyl flag too Uracil (U) - NO methyl flag, for RNA

A purine sites

When purines fall spontaneously off their sugars, so there's a blank space where there should be an actual base