DNA Replication and the Genetic Code (Midterm 3 Lesson 1)

what is the cell cycle

an alternation between interphase (normal cellular activity) and mitosis (cellular division)

• some terminally differentiated cells (eg mature neurons) stop dividing and enter G0 stage

when can you “see” chromosomes

right before cell division

what are the 3 possible models of dna replication

1. Semiconservative

2. Conservative

3. Dispersive

explain the semiconservative model of dna replication

it is the watson-crick model (made by the guys who first proposed the double helix model)

• each new DNA molecule consists of:

  • One original (parental) strand

  • One newly synthesized strand

  So after replication, each daughter DNA molecule is half old and half new — hence the term "semi" conservative.

explain the conservative model of dna replication

parental double helix remains intact, both strands of daughter helicies are newly synthesized

explain the dispersive model of dna replication

both strands of daughter helicies contain og and newly synthesized dna

explain the Meselson-Stahl experiment

grew e.coli with diff isotopes of nitrogen (N14 and N15)

• bacteria incorperate that N into dna (into nitrogenous bases)

• isolated dna after diff numbers of cell divisions

• detected isotopes based on density (centrifugation)

  • when N14 is put through centrifuge, the band created is up high (light), N15’s band is low (heavy)

• put the N15 dna strands in N14 medium (so any daughter dna would be N14 unlike parent N15 strand)

  • after first generation, new double helices were all in middle of N14 and N15 bands → showed double helicies were a mixture

  • after second gen, saw there were some strands that had ONLY N14 in them (some strands located up higher than the mixed ones) → shows they’re not dispersive or all dna would be a mixture

• confirmed dna replication is semi-conservative

how did the meselson-stahl experiment show that the new dna produced was semiconservative and not dispersive

bc 2nd gen had dna of ONLY 14N medium (one parental medium)

which bands would you expect to see from the meselson-stahl experiment if replication was conservative or dispersive

conservative = 2 diff bands in tube (N14 and N15) all throughout replications

dispersive = only one band in middle of 14N and 15N (everything would be mixed)

which direction does DNA synthesis go in

5’ to 3’

what direction is the template strand of dna read in

3’ to 5’

where are new nucleotides added on the growing strand

to the 3’-OH of the growing strand

which direction does DNA polymerase build new dna molecules in

5’ to 3’ direction

what are the 3 requirements for DNA pol activity? explain each

1. Four dNTPs (di nucleotide triphosphates)

   • get incorporated into growing chain

     • need the 4 (A,C,T,G) to properly build

   • cleaving of phosphate bonds provides E for dna pol activity

2. Single-stranded template (needs something to copy from)

   • other enzymes unwind a dsDNA (double stranded dna) molecule to exposed ssDNA (single stranded dna) segments

3. Primar with exposed 3’-OH end

   • dna pol cannot start a new strand → can only add nucleotides to existing strand

what are the 2 stages of dna replication

1. initiation

2. elongation

provide an overview of initiation in dna replication

enzymes open the double helix

provide an overview of elongation in dna replication

enzymes connect correct sequence of nucleotides on newly formed dna strands

• E for dna synthesis comes from high-E phosphate bonds in dNTPs

  • dna pol is the enzyme that catalyzes new phosphodiester bonds

explain the steps for initiation of dna replication in prokaryotes

1. initiator protein binds to origin of replication

   • origin of replication = short sequence of specific nucleotides (where initiator protein binds)

2. DNA helicase unwinds helix

   • replication bubble forms w a replication fork at each end (replicaton proceeds along the fork)

3. single-stranded binding (SSB) proteins keep dna helix open

   • and help stabilize the single-stranded dna

4. primase synthesizes rna primers

what do rna primers do

create an exposed 3-OH (hydroxyl) end that can be added onto by dna pol in dna replication

what is rna? how does it compare to dna?

ribonucleic acid

• less stable than dna

  • sugar in rna is ribose instead of deoxyribose

  • uracil is used instead of thymine

• can form a double-strand with DNA OR RNA through hydrogen bonding

what does dna polymerase 3 do

adds nucleotides to rna primer to make dna

which dna sequence is the nucleotide sequence in the offspring dna copied from

the template strand

which type of synthesis does the leading strand have in dna replication

continuous synthesis

which type of synthesis does the lagging strand have in dna replication

discontinuous synthesis

what are okazaki fragments

short dna fragments of the lagging strand

what does dna pol 1 do

replaces rna primer with dna nucleotides

explain the steps in elongation in dna replication in prokaryotes

reference…

• dna ligase

• leading strand

• template strand

• lagging strand

• dna pol 3

• dna pol 1

• okazaki fragments

• rna primers

1. nucleotides are added to rna primer to synthesize a new strand of dna (adding)

   • catalyzed by dna pol 3

     • adds nucs to rna primer to make dna

   • nucleotide sequence is copied from the template strand

   • leading strand has continuous synthesis in the direction of the replication fork (produces a 5’ to 3’ strand)

   • lagging strand has discontinuous synthesis to produce a 3’ to 5’ strand by synthesizing short 5’ to 3’ fragments

     • called okazaki fragments

2. dna pol 1 replaces rna primer w dna nucleotides (so there’s no rna in the dna)

   • dna ligase covalently joins adjacent okazaki fragments to complete the lagging strand (so they’re not just chunks)

how does the replication fork move (reference leading and lagging strands)

in opposite directions

• the leading strand moves continuously from one rna primer

• the lagging strand is synthesized using repeated primers placed as the replication fork moves (has to wait for 5’ primers to be placed as the replication fork moves downstream and then replicates dna back towards the center of the replication bubble) towards middle of replication bubble

note: all dna is still synthesized in the 5’ to 3’ direction

what does dna topoisomerase do

relaxes supercoils in prokaryotes

• cuts back sugar phosphate backbone

what does dna ligase do

seals unwound broken dna strands in prokaryotes

which processes allow for dna replication occur in circular bacterial chromosomes

replication proceeds in 2 directions from a single origin of replication (dont forget, circular chromosomes)

• synthesis occurs in both directions until replication forks meet

• unwinding of dna created supercoiled dna ahead of the replication fork (basically super tangled)

  • would stop pol from being able to transcribe

• dna topoisomerase relaxes supercoils

  • does so by cutting super phosphate backbone

  • causes dna strands to unwind

• unwound broken strands are sealed tg again by dna ligase

• dna replication occurs bidirectionally

• completes when replication forks meet at the termination region (last point of contact btwn the 2 dna molecules)

• topoisomerases separate the entwined daughter chromosomes, making 2 daughter molecules

what do exonucleases do

removes nucleotides

• double checks to make sure proper nucleotides are being added to new dna molecules, if not then exonucleases them (takes them off)

what do polymerases do

make polymers (eg dna)

what does proofreading mean in dna synthesis

means it can detect if the wrong nuc was added to the strand → minimizes mutations from occuring

what does dna polymerase 3 do in prokaryotes in regards to polymerase and exonuclease activity, along w their primary fx

major enzyme responsible for synthesis of new strands

• 5’ to 3’ polymerase actitivy

• 3’ to 5’ exonuclease activity, proofreading ability

what does dna polymerase 1 do in prokaryotes in regards to polymerase and exonuclease activity, along w their primary fx

responsible for removal of rna primers and gap filling

• 5’ to 3’ polymerase activity

• 3’ to 5’ exonuclease activity for proofreading

• 5’ to 3’ exonuclease activity for primer degradation

  • removes rna primers in 5’ to 3’ direction

what is the problem with humans having large chromosomes? what is our solution for this?

• you have to replicate all your dna in S phase

• would take too long if each chrom only had one origin of replication (and one replication bubble)

• to make it go faster, eukaryotes initiate dna replication at several points along the chromosome

t/f: prokaryotes have only one origin of replication

true (but eukaryotes typically have multiple)

what is the problem w having linear chromosomes

the removal of rna primers by ribonucleases results in newly synthesized dna strands being shorter (by the length of the rna primer → would eventually result in losing genes)

called the end-replication problem

how do eukaryotes combate linear chromosome depletion

telomeres

what are telomeres

regions of repeated non-coding dna at end of linear chromosomes

• contains repeated units of TTAGGG * 250-1500 (in humans, they’ll vary across species)

• non-coding regions of dna that can be depleted by the removal of rna primers so when the chroms shorten that shortening doesn’t eat into the acc dna

  • shorten in each cell division

  • “absorbs” the loss so that critical info (genes) are not immediately affected

what is telomerase and how does it work

has an rna sequence that compliments the telomere sequence

• EXTENDS/MAINTAINS TELOMERES

  • parent dna strand is elongated using complementary telomerase sequence

  • primase and dna pol synthesize remainder of complimentary strand

in other words…

• Telomerase is a special enzyme that extends the ends of linear chromosomes — specifically, the telomeres, which are repetitive DNA sequences at the tips of chromosomes

• Each time a cell divides, DNA polymerase cannot fully replicate the very end of the lagging strand (this is known as the end-replication problem). Without telomerase, telomeres would get shorter with every cell division — eventually leading to cell aging or death

note: telemerase is not in all cells (mostly just germ and stem cells ; it is not located in somatic cells → they j die when their telomeres are gone)

which dna strand is synthesized by primase and dna pol

the remainder of the complementary strand (the newly created strand)

what does telomerase consist of

a protein component and rna component

explain how telomerase works (reference telomerase elongation and translocation

translocation: protein in telomerase brings rna component to telomere where it binds to telomere sequence on the old strand of dna (leaving the new strand of dna w an exposed overhang)

elongation: exposed dna sequence has nucleotides bind to it and extend the complimentary dna strand

• makes it so primers will bind to telomere and THOSE regions of dna wont be transcribed but the coding regions will be

process repeats

what happens to telomerase after several rounds of elongation

• dna pol gap-fills (completes the other (new) strand so it doesnt just have nothing there)

  • from 5’ to 3’ end using primer on far side of the telomerase

• rna primer is degraded

• dna overhang is degraded

which cells in humans express and dont express telomerase

• reproductive cells do and a few kinds of stem cells do

• adult somatic cells dont

what is senescence

the eventual information (gene) loss in chroms with continuous cell division (degradation with age) → happens at less than 50 generations in a culture

what happens to animals w out any telomeres left on their chroms (even if they are the same age)? what does reintroducing telomerase do

• look much older

• have shorter life span

reintroducing telomerase spurs almost a complete recovery

BUT re-activating it in somatic cells greatly inc risk of cancer

• telomeres shortening limits amount of times cells can divide

  • w out that the cell can divide a lot more than usual

    • inc chances mutations will occur

how does dna lead to gene products

transcription and translation

what does transcription do

creates rna from dna

what is an rna transcript

output of transcription

serves directly as mRNA in prokaryotes; processed to become mRNA in eukaryotes

what does translation do

creates protein from rna

what is the diff between dna and rna

dna:

• deoxyribonucleic acid

• usually double stranded sugar phosphate

• contains nucleotide/nucleobase thymine

rna:

• ribonucleic acid

• usually single-stranded sugar phosphate

• contains nucleotide/nucleobase uracil

which molecules are these? say which is which specifically

what is this

deoxyribose (on rna)

what is this

deoxyribose (found on dna)

does deoxyribose or ribose have the shorter lifespan? what about between uracil and thymine

ribose and uracil have a shorter half-life than deoxyribose and thymine of dna

what is the primary structure of proteins

the AA sequence

what is the secondary structure of proteins

local folding

what is the teritary structure of proteins

overall shape of chain (including beta pleated sheets and alpha helices and stuff)

what is the quaternary structure of proteins

multiple chains tg

what determines the AA sequence in proteins

the rna sequence

how rna bases read (not direction, method)

in groups of 3 AAs (called codons)

how many possible values are there for each of the 3 bases individually in any individual codon

4 (one for each base in rna)

how many possible codons are there

4×4×4 (4 possibilities for each base, 3 bases in codon) = 64

what is AUG

the start codon → first codon translated into an AA

what are the stop codons

UAA, UAG, UGA (can check data sheet on test)

what is the template strand of dna

the dna that will actually bind / create base pairs with AAs from newly synthesized rna molecule

what is the antisense strand

the template strand of dna

what is the sense strand/coding strand of dna

the strand of dna that was previously bound to the template → will be identical to the new RNA strand produced (except T→U)

which direction is DNA read in translation and is mRNA read in translation

DNA is read in 3’ to 5’ to synthesize new mRNA strand from 5’ to 3’

mRNA is read from 5’ to 3’ direction and synthesizes new protein in that direction too (even though no 5’ or 3’ on new protein)

t/f: all rna molecules start with AUG

false → that is where the reading frame starts

what is the reading frame in translation

rna molecule does not start with AUG → the reading frame is from AUG to a stop codon

note: stop codon must be IN FRAME with the start codon

how do we know each nuc is part of only one codon (explain the observation and conclusion)

observation: each point mutation (mutation where one nuc is swapped for another) affects only one AA

conclusion: each nuc is part of only one codon

• if it was part of multiple then would have multiple protein mutations per point mutation

what are frameshift mutations and what are some examples of frameshift mutations?

mutations that change the reading frame of rna from then-on

• changes AA sequence

eg deletion and insertion

what is intragenic suppression

restoration of gene fx by one mutation cancelling the other in the same gene (eg equal # of insertions and deletions or 3 insertions/3 deletions) how did peole know which codon codes for w

how do we know that codons are 3 nucs long

3 single based deletions or 3 single base insertions result in frame restoration → example of intragenic suppression

how did people figure out which codon codes for which AA

added artificial poly-U-mRNAs to cell-free translation systems and tracked AA in polypeptide product

• added make mRNA with a bunch of radioactive Us into an environment where they could be translated and tracked the AA product

did this for all types of rna bases (all 4)

then introduced another base into the culture → could now have multiple products depending on the order of the 2 bases (eg Ser or Leu for UC) → made them alternate (UCUCUC)

• “charged” tRNA w either radiolabeled serine or leucine and non-radiolabeled of the other AA

• could then see which AA was added by if the complex was radiolabeled or not

explain the relationships of polarities in DNA, mRNA, and polypeptides

note: moving from 5’ to 3’ of each mRNA, each successive codon is interpreted into AA starting w N-terminus and ending w C-terminus

t/f: the genetic code is universal

false → it is ALMOST universal across species