Class 3: Molecular Biology (see notes)

Wrap DNA around histones

(-)DNA gets wrapped around (+)histones; the histone amino acids will be (+) charged like Lys, Arg so they attract (DNA+histones = nucleosomes) Most Condensed: Chromosomes Chromatin Nucleosomes Histones (8 histones make a nucleosome) DNA and sugar phosphate backbone

composite transposon

- has two IS elements and intervening sequence

DNA POL IV and V

- have similar characteristics - error prone in 5' to 3', but function to stall other polymerase enzymes at replication forks when DNA repair pathways have been activated

frameshift mutation

- insertions and deletions changes the reading frame, causes big changes in DNA (all amino acids change down the line) *not as bad if you insert/delete 3 codons or insertion/deletion comes towards end of strand

Polymerization occurs in the 5' to 3' direction, without exception

- the existing chain is always lengthened by the addition of a nucleotide to the 3' end of the chain - there is never 3' to 5' polymerase activity

transciption: template strand =

template strand = anti-sense strand, noncoding strand, transcribed strand - complementary to new mRNA

central dogma

theory that states that, in cells, information only flows from DNA to RNA to proteins - stepwise process reduces likelihood of DNA getting damaged; making copies keeps DNA authentic DNA = strings of nucleotides RNA = strings of nucleotides (same information as DNA) Proteins = amino acids (codons; 3 nucleotides = 1 codon = codes for amino acid) *4 different nucleotides to combine to form codons* *3 positions for those nucleotides* - 4 possible nucleotides in 1st position, 4 possible nucleotides in 2nd position, 4 possible in 3rd position = 4 x 4 x 4 = 64 possible codons you can create from nucleotides (ATCG) - 3 stop codons = 61 possible codons for amino acids (only 20 aa)** - 3rd position of codon doesn't usually matter (CUX codes for Leu no matter 3rd)

key info about prokaryotes

theta replication (1 chromosome) 1 origin (euk have multiple) genome is a single circular piece of dsDNA 5 different DNA polymerases (but mainly use III, I, II) 1 RNA polymerase no mRNA processing polycistronic mRNA - can get multiple proteins from one mRNA simultaneous transcription/translation smaller ribosomes (30, 50, 70S)

DNA and RNA are called nucleic acids because

they are found in the nucleus and possess many acidic phosphate groups - building block of DNA = deoxyribonucleoside 5' triphosphate (dNTP; N represents one of the four basic nucleosides)

Transcription diagram

**Transcription is the primary point of regulation for protein synthesis/translation** - RNA first binds to DNA at promotor region - start site is downstream from promotor - RNA POL first binds to promotor, cruise along DNA until it gets to start site where it will begin to start transcribing RNA - will keep doing this until it gets to STOP site: euk. = poly-A tail on DNA (made via poly adenylase enzyme) prok. = different sequence *RNA POL reads template strand in 3' to 5' direction and while doing so synthesizes mRNA in 5' to 3' direction Regulation: 1. promotor - binding site for RNA polymerase strong promotor: high affinity for RNA pol, lots of RNA transcribed weak: low affinity for RNA pol, less RNA transcribed *promotor = a sequence within DNA, hard to change how much protein is made if you have a weak one 2. DNA binding proteins: - repressors - bind to DNA to prevent transcription - enhancers - bind activators, that when brought close to promotor and other transcription factors, increase transcription *and operon/operator lies in between promotor and start site

Pyrimidines and Purines

- 2H bonds - 3 H bonds

tRNA structure

- 3' end = amino acid attachment site (top) - anticodon loop is on bottom end and contains 3 base region (anticodon base pairs with mRNA codon) ex: tRNA anticodon = UAC = tRNA-met mRNA codon = AUG so Met aa would attach at 3' end (Met-tRNA-met = aminoacyl-tRNA which is carried out by amino-acyl synthetase) *There are at least 20 tRNA, at most 61 (61 different codons), but can get by with fewer than 61 because wobble pairing - there are at least 20 tRNAs (20 aa) and less that 61 - as many aminoacyl tRNA synthetases as tRNAs *takes 2 ATP to attach amino acid to aminoacyl-tRNA*

DNA POL II

- 5' to 3' polymerase activity - 3' to 5' exonuclease proofreading function - participates in DNA repair pathways - backup for DNA POL III

wobble hypothesis

- Ability of the tRNAs to recognize more than one codon; the codons differ in their third nucleotide. - adenine can on tRNA can get converted to inosine (I) which allows for more flexibility - only expect to find inosine in tRNA not mRNA or rRNA Wobble base pairing happens when there is G, U, or I at the 5' end of the anticodon G -> C or U U -> A or G I -> A, U, or C *the same tRNA can be used to translate two different codons specifying the same amino acid; this is why you don't need 61 tRNAs C -> only G A -> only U

Which of the following produces a strand of DNA in the 5' to 3' direction? I. Eukaryotic DNA polymerase II. Prokaryotic DNA polymerase III III. Reverse transcriptase

- All three polymerases listed use a template strand to create a new strand of DNA. They do so by adding new nucleotides to the 3' end of the new strand (hence, the strand grows in the 5' to 3' direction). - Reverse transcriptase differs from the DNA polymerases only in that is uses an RNA template instead of a DNA template to synthesis its new strand of DNA. However, it too adds new DNA nucleotides in the 5' to 3' direction.

If a nucleotide is deleted during the transcription process this event would most likely lead to: A. no change in the protein. B. the destruction of the correct reading frame. C. an mRNA without a cap. D. a DNA gene with the incorrect base pairs.

- Deleting a nucleotide during transcription would result in a serious mutation called a frameshift mutation, which would completely destroy the protein's amino acid sequence and structure (choice B is correct and choice A is wrong). - It is unlikely that it would cause the mRNA to not be capped, so choice C is eliminated. - Finally, removing a base from RNA would have no effect on the DNA, as DNA replication does not depend on RNA at all (eliminate choice D).

Which one of the following mutations would be most likely to convert a proto-oncogene into an oncogene? A. Silent mutation B. Missense mutation C. Nonsense mutation D. Deletion mutation

- In order for the mutation to have the described effect, it must modify the protein without completely eliminating it or destroying its effect. A missense mutation converts a codon for one amino acid into a codon for a new amino acid, resulting in a small change within the protein's primary sequence, and an alteration (but usually not a total elimination) of the protein's function (choice B is correct). - A silent mutation converts a codon for an amino acid into a new codon for the same amino acid and has no effect on the protein product (eliminate choice A). - A nonsterm-30ense mutation creates a stop codon out of an amino acid codon, resulting in truncation of the protein and (usually) a loss of its function (eliminate choice C). - A deletion mutation eliminates one or more base pairs, altering the reading frame and drastically changing the amino acid sequence of the protein (eliminate choice D).

A graduate student in a yeast lab that studies double-strand break (DSB) repair has a mutant strain that is unable to complete repair via nonhomologous end joining. Which of the following is true about this strain?

- It is able to form a joint molecule when repairing DSBs - This mutant strain is unable to complete nonhomologous end joining and is thus relying on homologous recombination for DSB. This is a specific repair process that involves formation of a joint molecule and uses DNA polymerase - A strain that repairs DNA via homologous end joining will be able to repair DSBs reasonably well, and will not accumulate many chromosomal aberrations over time

An error is made during production of an RNA transcript which results in a change in a single amino acid in the final protein. This error is most likely a result of: A. incorrect translation of the mRNA into protein by the ribosomes. B. the poor editing ability of RNA polymerase. C. a frameshift mutation occurring during transcription. D. incorrect translation of the DNA into RNA by RNA polymerase.

- RNA polymerases have no editing function since RNA is a transient molecule and its information is not passed on to offspring (choice B is correct). - The question states that the error is made during production of the RNA, so the change in amino acid sequence reflects the new nucleotide sequence and is not due to incorrect translation of the mRNA (choice A is wrong). - Frameshift mutations are very severe and would result in all of the amino acids from the error on being changed (choice C is wrong) - DNA is not translated by RNA polymerase, it is transcribed (choice D is wrong).

cDNA (complementary DNA) is often reverse transcribed from mature (processed) eukaryotic mRNA in order to create DNA without introns. The enzyme reverse transcriptase is used to create the initial complementary DNA strand, and requires a short sequence of DNA to act as primer. Which of the following sequences would make a good primer? A. Any random sequence of DNA could be used, as long as it has a free 3' end for reverse transcriptase to act on. B. Any random sequence of DNA could be used, as long as it has a free 5' end for reverse transcriptase to act on. C. UUUUUUUU D. TTTTTTTTT

- Since DNA and RNA polymerization always take place in a 5' to 3' direction, each polymerase needs a free 3'-OH group so polymerization can occur (choice B is wrong). Although a random DNA sequence might coincidentally match the mRNA somewhere in its sequence and bind there, a poly-T sequence would be guaranteed to pair with the 3' poly-A tail of the mRNA(choice D is a better answer than choice A). Uracil (U) is a component of RNA, not DNA (choice C is wrong).

Rad21 functions in double-strand break repair and is also a subunit of the chromatid cohesin complex, necessary to keep sister chromatids connected. Cells with non-functional Rad21 due to mutation will display all the following phenotypes EXCEPT: A. increased sensitivity to UV and reactive oxygen species. Your Answer B. decreased ability to maintain chromosomes in the 2x state. C. faster than usual mitotic phase due to increased chromosome motility. Correct Answer D. increased probability of cell cycle arrest at the G2/M transition.

- UV light and reactive oxygen species can both induce double-strand breaks in DNA. If cells have defective Rad21, they will have a diminished ability to repair these breaks (choice A is true and can be eliminated). - Since Rad21 is important in chromatid cohesion, without functional Rad21, the sister chromatids may not be able to remain joined after DNA replication. This means cells would revert to a 4n (tetraploid) 1x (one chromatid per chromosome) state instead of the 2n 2x state that is normal in G2 and the first part of mitosis (choice B is true and can be eliminated). - If double-strand break repair cannot occur to full capacity, the cell would likely arrest at the G2/M transition (choice D is true and is can be eliminated). - However, there is no reason why decreased double-strand break repair or poor chromatid cohesion would cause a faster mitotic phase or increased chromosome motility. If anything, mitosis would be arrested as the cell tried to deal with disorganized chromosomes (choice C is a false statement and is therefore the correct answer).

Nucleic acid polymerization - think about what joining a HPO4- group to an OH would do

- aka a condensation reaction or dehydration synthesis - dehydration synthesis = formation of new chemical bonds between two molecules which leads to the formation of new compounds. A reaction occurs with the loss of water molecule at each step. - 5' to 3' synthesis and base sequence - antiparallel and complementary - phosphodiester bond - phosphate is always added to 3' end reaction OPP to hydrolysis reaction = cleavage of chemical bonds by the addition of water or a base that supplies the hydroxyl ion ( OH−). A chemical bond is cleaved, and two new bonds are formed, each one having either the hydrogen component (H) or the hydroxyl component (OH) of the water molecule. - water is added to a substance

Telomeres

- another region on the chromosome 1. the ends of linear chromosome, made up of both single and double stranded DNA (base pairs with itself and leaves free end hanging) 2. stabilize the ends of chromosomes by capping them 3. consists of short nucleotide repeats (TTAGGG)

Transposons

- both prok and euk have these mobile genetic elements - 3 types: IS element, Complex Transposon, Composite Transposon IS element - composed of transposase gene flanked by inverted repeat sequences Complex - contains additional genes Composite - 2 transposons together (2 full transposase enzymes and inverted repeat sequences) flanking either side of a gene - have 2 similar or identical IS elements with central region in between - if same directions: 1. deletion 2. insertion - if opp directions: 1. intergenic region will be inverted 2. the 2 transposons are unaffected All transposons have gene that codes for protein transposase = cut and paste activity, where it catalyzes mobilization of the transposon (excision from the donor site) and integration into a new genetic location (the acceptor site) - if a chromosome has 2 transposons with inverted orientations, they can again pair and align with each other. After recombination, the sequence of DNA between the 2 transposons ends up inverted

complex transposon

- contains an IS element (the transposase and its accompanying inverted repeat sequences) and one or more genes

tRNA translation and energy count

- contains anticodon that mRNA will bind to - at least 20 tRNA and at most 61 - amino acyl tRNA synthatase has specific anticodon that binds aa - EPA sites located on large ribosome - first tRNA loads into P site - incoming tRNA enters A site and forms peptide bond with other amino acid - no tRNA that recognizes STOP codon, instead bind release factor - this breaks bond between final tRNA and final amino acid to release completed protein 2 ATP to load an amino acid (per tRNA) aka aa activation + 1 ATP to position AUG-start codon loaded onto P site (initiation) + 1 ATP to load another aminoacyl tRNA into A site + 1 ATP for translocation + 1 ATP for termination # amino acids x 4 = # of ATP needed

Translocations

- due to faulty DNA repair (non-homologous end joining) or abnormal recombination between non-homologous chromosomes

nucleic acid polymerization

- free 5'C - free 3' OH group where more nucleotides (base + sugar + phosphates) can be added to make a nucleic acid (=2+ nucleotides linked together) 5' to 3' synthesis and base sequence: - sugars (5C ribose or 5C deoxyribose) and phosphates = backbone of DNA molecule - the bases give the sequence (AGCUT) - to name base sequence first start with base that is attached to sugar with free 5' carbon, next is base attached to sugar with free 3' OH group antiparallel (strands of DNA run opposite to make double stranded) and complementary (bases AGCUT are paired complementary) nucleotides are held together with phosphodiester bond (one phosphate double bonded to an O and an O-R group x2) **3'OH is nucleophile that attaches phosphate, pyrophosphate is LG**

The inner region of DNA is

- hydrophobic - due to the nitrogenous bases with many carbon atoms and few polar groups The region surrounding DNA and the phosphate-ribose backbone of DNA are both hydrophilic; the hydroxyl groups on the ribose and the deprotonated hydroxyl groups on the phosphate allow this portion of the DNA molecule to readily interact with water

DNA Replication is semiconservative

- individual strands of the double-stranded parent are pulled apart, and then a new daughter strand is synthesize using the parental DNA as a template copy from. - each new daughter strand is perfectly complementary to its template or parent

Post-replication repair

- mismatch repair pathway (MMR) - a type of homology repair that happens DURING OR AFTER DNA REPLICATION

Euchromatin

- not rich in repeats - GAPDH is a housekeeping gene and is expressed continuously. A gene that is always turned on must be accessible to transcription factors. Only euchromatin is in a loose conformation and readily accessible for transcription. LOOSELY WOUND so transcription can occur - think cell cycle and mitosis PMAT

Eukaryotic DNA Replication

- occurs at many different points along eukaryotic chromosomes = multiple origins = replication can proceed at many different locations on the chromosome at same time - contain several DNA polymerases = multisubunit enzymes **occurs during S-phase of cell cycle**

Lac operon

- regulates synthesis based on cell needs (not just promotor) in bacteria - bacteria prefer using glucose as main E but sometimes must use lactose operon = promotor + all genes its controlling PROG - in absence of lactose, repressor is bound to operator, repressor prevents RNA POL from getting to start site to transcribe lactose genes = no synthesis of proteins - in presence of lactose, lactose binds to repressor which removes it from operator, RNA POL can now get to start site and transcribe lactose genes to digest it *steroid hormones work similar to this mechanism

DNA Pol III

- responsible for super-fast, super-accurate elongation of the leading strand - high processitivity - has 5' to 3' polymerase activity as well as 3' to 5' exonuclease activity (will cut a nucleic acid chain at the end) = proofreading function - it has no function in repair so it is simply a replication enzyme

Heterochromatin

- rich in repeats (centromeres and telomeres are made of heterochromatin) centromeres = the region of a chromosome to which the microtubules of the spindle attach, via the kinetochore, during cell division. (**Centrosomes are organelles which serve as the main microtubule organizing centers for animal cells.)

DNA Packaging Eukaryotes

- several linear chromosomes (so they have much larger genomes than prokaryotes) DNA (-) Histones (+) Nucleosomes (8 histones = octomer get wrapped around nucleosome = beads on a string) Chromatin Chromosomes inside nucleus (protects DNA) Region on the chromosome: 1. Centromere: where sister chromatids attach during mitosis, and where spindle fibers attach during mitosis = metaphase short arms (p) long arms (q) 1. euchromatin: lightly packed form of chromatin (DNA, RNA, and protein) that is enriched in genes, and is often (but not always) under active transcription. Euchromatin comprises the most active portion of the genome within the cell nucleus. 92% of the human genome is euchromatic 2. heterochromatin: tightly wound, inactive, dark staining

DNA Packaging Prokaryotes

- single circular dsDNA genome - all resides in cytoplasm so subject to damage from other molecules/chemicals/degradation via enzymes in the cytoplasm 1. methylation: protection from their own restriction enzymes (cut up viruses' DNA that are not methylated); methyl groups attached to DNA prevent restriction site from fitting into active site of enzyme 2. supercoiling: helps package genome to save space/protect it (DNA gyrase, a bacteria enzyme will be used to supercoil the genome to make it easier to store; prokaryotes have no nucleus to package genome - their genome is supercoiled in the cytosol. DNA gyrase creates ds-breaks in 5' to 3' phosphodiester bonds between nucleotides) *still has double stranded helix **doesn't contain telomeres because circular genomes

Prokaryotic DNA replication (aka Theta replication)

- working with circular dsDNA helix - 1 origin, where replication starts - 5 DNA polymerases: DNA POL III (main) - high processivity - fast 5' to 3' polymerase AND 3' to 5' exonuclease (backs up to cut a nucleotide at end of strand in DNA = proofreading function, must recognize mistake right away because not an endonuclease) - adds nucleotides at 400 bps downstream of ORI - no function in DNA repair (only proofreading, can't go back once enzyme is too far downstream) DNA POL I - low processivity - adds nucleotides at RNA primer (goes for about 300 bps then POL III takes over) - slow 5' to 3' polymerase AND 3' to 5' exonuclease (back up, proofreading) - also a 5' to 3' exonuclease to remove primer - DNA excision repair less important: DNA POL II: - 5' to 3' polymerase AND 3' to 5' exonuclease (proofreader) - back up for DNA POL III - DNA repair *many mutant cells that lack DNA POL II are able to survive = not critical to replication DNA POL IV and V: - error prone 5' to 3' activity - DNA repair

How transposons contribute to genetic variation

1. Code for the "cut and paste" transposase enzyme (transposase gets trasncribed and translated) 2. Transposase cuts transposon out 3. Transposase "pastes" transposon somewhere else Effect of a single transposon: If inserted in intergenic region: probably nothing bad happens - intergenic is not the same as intronic region (portion within DNA that is not usable); intergenic region = region in between 2 independent genes If inserted into a coding region: that gene won't work = mutation

DNA replication (4 rules and requirements for carrying out DNA replication)

1. Semiconservative = half of the original DNA molecule will be saved in the new DNA molecule 2. occurs in 5' to 3' direction (all nucleic acid synthesis occurs in this direction) 3. requires a primer (DNA pol cannot create a strand out of nothing, must provide existing strand for it to work on = piece of RNA primer that DNA pol elongates) 4. requires a template (existing form of nucleotides)

Posttranslational Modification Insulin Ex

1. insulin displays all 3 types of posttranslation modification: - proinsulin folding - proinsulin covalent modification (disulfide bonds) - proinsulin processing (cleave two portions of molecule which is called C-peptide) = mature insulin

Post-translational modification

1. protein folding - aided by chaperons 2. processing - cleavage to form activated protein (i.e zymogens) - occurs in cytoplasm - eukaryotes only 3. covalent modification (all below): - disulfide bridges - formylated (addition of a formyl group (-C(O)H) - alkylated (addition of an alkyl group (methyl, ethyl, etc). Methylation is a common post-transcriptional modification, and is usually done to lysine or arginine amino acids - glycosylation (addition of a glycosyl group to arginine, asparagine, cysteine, serine, threonine, tyrosine, or tryptophan; a glycosyl group is the substituent form of a cyclic mono-, di-, or oligosaccharide = this results in a glyoprotein) - phosphorylation (addition of a phosphate group (PO43-) to a serine, threonine, tyrosine, or histidine) - acetylated (addition of an acetyl group (-C(O)CH3) at N-terminus of protein or at a lysine amino acid) - sulfated (addition of a sulfate group (SO42-) to a tyrosine amino acid

Characteristics of RNA

1. single stranded, except in some viruses 2. Uracil 3. the pentose ring in RNA is ribose rather than a 2' deoxyribose - why the RNA polymer is less stable, because the 2' hydroxyl can nucleophilically attack the backbone phosphate group of an RNA chain, causing hydrolysis when the remainder of the chain acts as a LG - this cannot occur in DNA bc no 2' OH

triphosphate

3 phosphate groups

Mutation: Endogenous Damage

= DNA damage that comes from inside the cell: a. reactive oxygen species b. physical damage 1. oxidized DNA (bases can no longer base pair with one another, strands separate, polymerase can't recognize) 2. crosslinked bases (bases are physically linked together, not just H-bonded = can't separate DNA, or replicate) 3. double or single stranded breaks (poor replication after this) 4. these can lead to polymerase errors, or failure to replicate all together Repaired by: 1. nucleotide excision repair both of these are for if DNA is PHYSICALLY damaged: 2. homologous end joining or 3. non-homologous end-joining

Inversion/Deletion/Amplification (of transposons)

= addition of one or more extra nucleotides into the DNA sequence = removal of nucleotides from the sequence chromosome amplification = when a segment of a chromosome is duplicated/multiplied - this messes up HOW MUCH product you make, not the product itself, both products are regulated the same (because identical) but now you have 2x - 4x as much both cause a shift in the reading frame INVERSION moves segment of DNA to NON-HOMOLOGOUS CHROMOSOME

ATP

= adenosine triphosphate = ribonucleotide (ribose is the sugar, not deoxyribose) - contains phosphodiester anhydride bonds

Mutation: Exogenous Damage

= damage that is coming from outside the cell: a. radiation (UV, x-ray, chemical) b. chemicals 1. UV radiation = pyrimidine dimers (CUT pairs with each other) 2. x-rays = ds breaks and translocations 3. chemicals = can lead to physical damage to DNA or to intercalation and thus polymerase errors Repaired by: 1. direct reversal by white light (dimers) 2. homology-directed repair or non-homology end-joining 3. nucleotide excision repair

Telomerase Extension

= lengthens telomeres by adding repetitive nucleotide sequences (does this by binding TTAGGG sequence on telomere) characteristics: 1. built in RNA primer 2. reverse transcriptase activity

Monomers

= nucleotides/nucleosides (lack phosphate group) nucleosides: A-ribose = adenosine G-ribose = guanosine C-ribose = cytidine T- ribose = thymidine U-ribose = uridine *in a dilute solution A and T will not bind to each other but to water

Phosphate

= orthophosphate (= single phosphate; (PO4)3-) - two bound together via anhydride linkage form a pyrophosphate 3 reasons phosphate anhydride bonds store so much E: 1. when phosphates are linked together, their negative charges repel each other strongly 2. orthophosphate has more resonance forms and thus lower free energy than linked phosphates 3. orthophosphate has a more favorable interaction with the biological solvent (water) than linked phosphates

translocations

= recombination occurs between nonhomologous chromosomes placing previously unconnected sequences in proximity - can create gene fusion, where a new gene product is made from parts of 2 genes that were not previously connected - common in cancer - can be balanced = no genetic information is lost (probably homologous end joining) - or unbalanced = where genetic info is lost or gained (lost if its non-homologous end joining) **can occur due to faulty DNA repair (i.e from non-homologous end-joining) or abnormal recombination between non-homologous chromosomes**

Mutation: (DNA) Polymerase Errors

= typo/small scale mutations 1. point mutations (polymerase makes a mistake, single base pair changes; doesn't get corrected on proof reading so remains as a mutation) 2. small repeats (polymerase slips off DNA strand, rejoins a few bases down and replicates a few bases making repeats) 3. insertions/deletions (small, frameshift; could be due to chemicals) Repaired by: 1. mismatch repair pathway (after replication; polymerase made a mistake and didn't catch it) 2. nucleotide excision repair (before replication; damaged base needs to be removed and insert correct base before DNA strands are replicated)

Point mutations

A -> G = a single base pair change transitions = purine for purine transversions = purine for pyrimidine missense = amino acid replaced for new aa, may not be serious if exchange is with similar amino acids nonsense = codon for aa becomes a STOP codon (shorted protein) silent mutation = a codon is changed to a new codon for the same amino acid (no effect); may see a change at the phenotypic level if translation is paused for translating a rare codon (may get less of protein due to time) that was switched for a common one and vice versa

start codons

AUG - required to start translation

A biologist designs a fluorescent form of DNA helicase which emits visible light at the edge of spreading replication forks. She then images an unknown cell and observes several dozen fluorescent puncta. Which of the following best characterizes the cell and why? A. Prokaryotic: multiple locations of active replication are visible on a single chromosome. B. Prokaryotic: multiple locations of active replication are visible on multiple chromosomes. C. Eukaryotic: multiple locations of active replication are visible on a single chromosome D. Eukaryotic: multiple locations of active replication are visible on multiple chromosomes.

D The researcher observes several dozen fluorescent puncta which would indicate several dozen replication forks. Prokaryotic cells only possess a single origin of replication and would only display two replication forks and therefore two puncta (choices A and B are wrong). Eukaryotes possess multiple chromosomes with multiple origins of replication on each chromosome; many more replication forks would be observed during replication (choice D is correct and choice C is wrong).

Prokaryote polymerases

DNA POL III - no known function in DNA repair, high processtivity, fast 5' to 3' polymerase and 3' to 5' exonuclease activity (editing tool, can replace wrong bases), MAIN REPLICATING ENZYME DNA POL I - DNA excision repair, low processitivty, slow 5' to 3' polymerase and 3' to 5' exonuclease activity, also has 5' to 3' exonuclease activity to remove RNA primer, adds nucleotides at RNA primer DNA POL II - back up for DNA POL III, 5' to 3' polymerase and 3' to 5' exouclease, DNA REPAIR DON'T USE THESE AS MUCH: (DNA POL IV and V - error prone 5' to 3' polymerase activity, DNA REPAIR)

Other important enzymes for DNA replication

DNA replication: - helicase (unwinds DNA at ORI) - topoisomerase (cuts on or two DNA strands to prevent supercoiling from helicase) - SSBPs (= protect DNA that has been unpackaged in preparation or replication and help keep strands separated) - primase (=RNA polymerase that lays primer, ligase, telomerase at origin) - DNA Polymerase (binds RNA primer and elongates it. A multi subunit enzyme. Will also proofread and remove RNA primer) - DNA ligase (links Okazaki fragments and larger leading/continuous strands) Transcription: spliceosome machinery Translation: aminoacyl tRNA, synthetases, initiation factors, elongation factors, release factors

Read direction

DNA replication: 3' to 5' on the DNA template Transcription: 3' to 5' on the DNA template Translation: 5' to 3' on the RNA template

Build direction

DNA replication: 5' to 3' (requires a primer) = slightly slower because editing exists Transcription: 5' to 3' (RNA POL doesn't require a primer to start) = faster mechanism because no editing here; RNA is a transient molecule Translation: N-terminus to C-terminus

Template molecule

DNA replication: DNA Transcription: DNA Translation: mRNA

Molecule synthesized

DNA replication: DNA Transcription: RNA (mRNA in prok, hnRNA in euk) Translation: peptides

Key synthesis enzyme

DNA replication: DNA POL Transcription: RNA POL Translation: ribosome (made of rRNA and peptides)

Signal to get ready

DNA replication: ORI Transcription: promotor Translation: Shine-Dalgarno (prok), Kozak sequence (euk), these are found in 5'UTR

Signal to start

DNA replication: ORI (start site); requires DNA template Transcription: start site (no primer required), requires DNA template Translation: AUG start codon

Prokaryotic location

DNA replication: cytoplasm Transcription: cytoplasm - Because transcription in prokaryotes occurs in the cytoplasm, ribosomes are able to bind and begin translation even before transcription is complete; prokaryotic mRNA requires no additional processing after transcription; they lack a mechanism for splicing out introns.(bacteria would not likely be able to produce a large gene from eukaryotes if it requires the splicing mechanism) Translation: cytoplasm

Eukaryotic location

DNA replication: nucleus Transcription: nucleus Translation: cytoplasm

Signal to stop

DNA replication: when the replication bubbles or newly synthesized strands meet and are ligated together Transcription: transcription stop sequence or poly-A sequence Translation: stop codon (UAA, UGA, UAG)

DNA vs RNA

DNA: - ds - thymine - deoxyribose (2'H) - double helix - one type RNA: - ss - uracil - ribose 2'OH) - lots of different shapes - several types 3 Primary types of RNA rRNA (RNA POL I) = Non-Coding RNA mRNA (RNA POL II) = only Coding RNA tRNA (RNA POL III) = Non-Coding RNA Others: hnRNA - heterogenous nuclear RNA (not modified at all) miRNA and siRNA (microRNA and small interfering RNA) - prevents mRNA from doing its job

DNA in the nucleus

Deoxyribose -> add base (AGCUT) -> nucleoside -> add 3 phosphates -> nucleotide -> polymerize with loss of 2 phosphates -> oligonucleotide -> continue polymerization -> single-stranded polynucleotide -> two complete chains H-bond in antiparallel orientation -> ds DNA chain -> coiling occurs -> ds helix -> wrap around histones -> nucleosomes -> complete packaging -> chromatin

Eukaryotic DNA Replication: End of Chromosome

End Replication Problem - RNA primers add at lagging strand - RNA primers removed - lagging DNA strand is shorter than leading strand = shorter telomeres (end of chromosome) - every round of replication shortens the telomeres - eventually, telomeres will shorten so much that you run into protein coding regions, cell won't be functional (this is combatted via telomerase enzyme; some cancer and stem cells have)

phosphoric acid

H3PO4= an inorganic acid (does not contain a carbon) with the potential to donate 3 protons - the pKa for the 3 acid dissociation equilibria = 2.1, 7.2, 12.4 - therefore, at physiological pH, phosphoric acid is significantly dissociated, existing largely in anionic form - the most common species (60% in extracellular fluid) = hydrogen phosphate: (HPO4)-2 - the second most common = dihydrogen phosphate: (H2PO4)-

HDACs change chromatin by: increasing its condensation and inhibiting transcription.

HDACs counter the effects of histone acetyltransferases (HATs). As histone acetylation typically promotes transcription by modifying chromatin structure, HDACs would inhibit transcription by condensing chromatin structure.

Direct reversal by white light (dimers) - is this found in humans?

Mutation: Exogenous damage (UV radiation/x-rays) Direct reversal = no extensive complicated pathway. - visible light reverses dimerization (C=C, U=U, T=T) - not found in humans - similar to nucleotide excision repair - photoreactivation - when UV creates a pyrimidine dimer in dsDNA, white light is used - photolyases activated by high frequency light and they break the covalent bonds of the pyrimidine dimer

Non-homologous end joining

Mutations: Endogenous damage - repairs dsDNA breaks - no sister chromatid for template (cell is not going through division and thus no replicating DNA) - MUTAGENIC because losing some bases, or could result in a translocation (wrong chromosomes connected) - still better than having a break in strand A quick-and-dirty mechanism for repairing double-strand breaks in chromosome/DNA that involves quickly bringing together, trimming, and rejoining the two broken ends; results in a loss of information at the site of repair. - no "check in" mechanism to make sure you are putting the correct pieces together

Homologous end joining repair

Mutations: Endogenous damage - repairs dsDNA breaks - sister chromatids can be used as a template to repair broken strand - happens after replication (requires sister chromatid as template) - typically only in meiosis (basically only in gametes) - uses homologous chromosomes (copy you get from parents) as a template to fix the broken one - can be divided into repair that happens before DNA replication (excision repair) or repair that happens during and after DNA replication (post-replication repair) ex: gene on chromosome 2 from mom would be replaced by chromosome 2 from dad if there was damage = homologous chromosomes X X or x x not sister chromatids X or non-homologous chromosomes: Xx

Mismatch Repair Pathway

Mutations: Polymerase errors - detected after replication is complete; polymerase made a mistake and didn't catch it - this pathway repairs bases due to DNA polymerase errors - recognize mismatch, determine which side is more methylated (will be older parent strand), so new daughter strand with mismatch can be identified - cut out a piece of newer strand, replace using DNA pol

Base/Nucleotide Excision Repair

Mutations: Polymerase errors - a type of homology repair that happens BEFORE DNA REPLICATION because defective bases will lead to polymerase errors - simply remove bad base and replace (before polymerase gets there) - base excision repair cuts out and replaces damaged bases - nucleotide excision repair identifies and replaces bulky deformations of dsDNA (ex: pyrimidine dimers)

pyrophosphate

P2O7 4- (2/3 phospates must come off a dNTP before it can join another dNTP via phosphodiester linkage in the 5' to 3' direction)

Source of mutations (4)

Polymerase Errors Endogenous Damage - caused by something inside the cell Exogenous Damage - damage to DNA caused by something outside the cell Transposons

Prokaryotic vs Eukaryotic Transcription

Prok: 1) Transcription and translation happen in the same place, at the same time (cytosol) 2) no mRNA processing: mRNA is ready to go 3) polycistronic: several different proteins can be translated from the same piece of mRNA 4) use single RNA polymerase Euk.: 1) Transcription in nucleus, translation in cytosol - cannot occur at same time due to this - also hnRNA from nucleus is not mRNA right away and must be processed before being translated (5' G-cap, 3' poly A tail, splicing) 2) monocistronic = one mRNA, one protein 3) have 3 different RNA polymerases: - RNA POL I transcribes rRNA - RNA POL II transcribes mRNA - RNA POL III transcribes tRNA

DNA POL I

Starts adding nucelotides at the RNA primer, poor processivity so usually taken over by DNA pol III about 400 bp downstream from the ORI, also capable of exonuclease activity, removes the RNA primer via 5' to 3' exonuclease activity, leaving behind DNA in 5' to 3' activity, important for excision repair

Post-transcriptional modification

The enzymatic processing of the eukaryotic primary RNA transcript to produce a mature transcript; production of a mature mRNA requires 5' capping, 3' polyadenylation and intron splicing. - happens in nucleus - eukaryotes only hnRNA remains in nucleus until modifications are made and then mRNA moves into cytoplasm

amber codon

The triplet UAG, one of the three termination codons that end polypeptide translation = a stop codon (for example TAG)

stop codons

UAG UGA UAA

methylation

decreases transcription; decreases DNA expression

Other types of RNA

hnRNA = heterogeneous nuclear RNA, initial unprocessed transcript (first piece of mRNA synthesized during transcription in eukaryotes, still needs to be processed: tail, cap, introns) miRNA and siRNA = micro RNA and small interfering RNA, help regulate gene expression (miRNA can bind to mRNA in cytosol = double stranded and now this cannot be translated)

acetylation

increases transcription; increase DNA expression

Mutation: Transposons

jumping genes 1. insertions/deletions (large) 2. chromosomal inversions 3. chromosomal duplications Repaired by: - generally don't lead to repair mechanisms because chromosome doesn't actually look damaged (is longer/shorter)

Transcription =

primary point of regulation for translation Regulation: 1. Promotor - strong = high binding affinity to RNA POL = higher expression of gene - weak = low affinity for RNA POL = low expression of gene 2. DNA binding proteins - silencers = repressors = can turn off a gene or repress it - enhancers = can enhance/turn on a gene

Adenine

purine - connects to thymine - bases held together by 2 hydrogen bonds

Guanine

purine - connects to cytosine - bases held together by 3 hydrogen bonds (must heat to a higher temperature to separate strands)

Thymine

pyrimidine - connects to adenine - 2 hydrogen bonds

Uracil

pyrimidine - replaces thymine for RNA - uracil can do 3 H bonds but adenine can only do 2

3 Primary types of RNA

rRNA = ribosomal RNA, catalytic part of functional ribosome mRNA = messenger RNA, sequence of codons determines amino acid sequence of protein tRNA = transfer RNA, carries amino acids to ribosome

key info about eukaryotes

replication bubbles multiple origins (because each chromosome has one) genome is several linear pieces of DNA 3 DNA POL (but only use DNA POL III) 3 RNA POL (I-rRNA, II-mRNA, III-tRNA) capping, tailing, and splicing of mRNA prior to translation monocistronic mRNA (one mRNA = one protein) transcription in nucleus translation in cytosol larger ribsomes (40, 60, 80S)

transcription: coding strand =

sense strand - not being transcribed - will have same sequence as mRNA except for Us and Ts

Telomere Extension (eukaryotes)

think TTAGGG sequence - elongates telomeres on parent strand of DNA 1. has a 9 base long RNA template 2. but adds 5'-TTAGGG-3' repeats (about 50-100 of them) Cells that express telomerase: - should only be active in stem cells but some cancer cells might have active telomerase