DNA: Chapters 16-18, 20Choose a topic: Structure

Replication

Mutations

Transcription/Translation

Gene Expression

Other Technologies

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Structure

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• Double helix made up of 4 bases; 2 purines that are made up of 2 rings (adenine, guanine), and 2 pyrimidines that are made up of 1 ring (thymine, cytosine)

– A-T with 2 hydrogen bonds, G-C with 3 hydrogen bonds• Double helix has 10 nucleotide (NT) pairs per twist and keeps a constant diameter of 2nm• DNA always built in the 5’-3’ direction with the phosphate attached to the 5’ carbon of the sugar on one NT binding to

the 3’ carbon of the sugar of the next NT– Makes up the sugar phosphate backbone

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sugar

phosphate

base

Packaging

• DNA double helix has a diameter of 2nm– Wrapped around histones, make up nucleosomes

referred to as “beads on a string”. Amino end of histone extends outwards (histone tail) - 10nm

• The protruding histone tails interact and link together causing the 10nm to coil and fold condensing to 30nm, common during interphase– The 30nm fiber will loop into looped

domains attached to a “protein scaffold” and condenses to a diameter of 300nm» Looped domains further coil into

a metaphase chromosome 700nm in diameter

• During interphase chromatin still sometimes condensed into heterochromatin vs. the less condensed euchromatin

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Replication• DNA replication is guided by enzymes that are grouped together in a replisome/DNA replication complex• Steps of DNA replication

– Replication starts at specific origins of replication, marked by specific sequences. Helicase opens the double helix, separating the parent strands, single strand binding proteins temporarily prevent repairing of base pairs

– Primase creates a short sequence of 5-10 RNA nucleotides (NT) called a primer. DNA polymerase will build 5’-3’ – DNA polymerases require a primer and a template strand

1. Complementary bases added to the template (parent) strand, elongating the primer. NTs come from nucleotide triphosphates that lose phosphates when added, release energy to drive the process

– Elongating towards the fork, polymerases added continuously, adding to the leading strand that will require only 1 primer, opposite direction (lagging strand) must keep restarting with multiple primers, make up Okazaki Fragments 100-200 NTs long that are formed by DNA Polymerase III, they are later joined together by DNA Ligase and RNA primers are replaced with DNA nucleotides by DNA polymerase I

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After Replication

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• DNA polymerases check for errors, if this “proofread” misses errors, enzymes will swap in correct bases in process called mismatch repair before the errors become permanent mutations

– Still damaged segments cut out by nuclease and filled back in with correct bases by polymerases using the template strand

• Because strand only elongates 5’-3’, no way to repair the 5’ end, buffered by telomeres, sequences repeated 100-1000 times that become shorter as cells divide but protect coding sections.

– Degeneration of telomeres contributes to aging, telomerase in germ cells continually elongates telomeres so that gametes will not eventually be without genes

– Telomerase found to contribute to cancer, active in tumors allowing uninhibited division

Mutations• Point Mutation=changes in single NT pairs, can have small or large impacts

– 3 types of point mutations:• Subsitutions = replacement of single NT pair with another pair• Insertions/deletions = +/- of NT pairs• Frameshift mutations alter the reading frame and thus change all subsequent codons

– 3 categories of results from a mutation:• Silent Mutations have no effect on phenotype• Missense Mutations change what amino acid the codon codes for• Nonsense Mutations change the codon to a stop codon resulting in premature termination of the

polypeptide chain• Common causes of mutations are:

– X Rays– UV Radiation can cause thymines to bind to each other causing the DNA to buckle - thymine dimers are

permanent damage

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Transcription• Transcription = synthesis of RNA from DNA, RNA for a protein coding gene =

messenger RNA (mRNA)– Yields primary transcript (pre-mRNA) in eukaryotes that needs processing

before being usable• Prokaryotes primary transcript requires no modification before translation• RNA Polymerase can work without a primer, attaches at a promoter

(includes start point for transcription) and stops at a terminator, stretch of DNA being transcribed is the transcription unit

– Initiation:• In prokaryotes, RNA polymerase binds by itself, in eukaryotes, it will not

bind until transcription factors have bound to the promoter– Polymerase + transcription factors = transcription initiation complex– TATA box = sequence commonly found in eukaryotic promoters

– Elongation:• RNA polymerase moves down the template strand untwisting 10-20 NT at

a time, building 5’-3’, 40 bases/sec in eukaryotes– Termination:

• Prokaryotes: transcription proceeds through a terminator sequence that tells polymerase to detach and the strand needs no further modification

• Eukaryotes: RNA Polymerase II transcribes a polyadenylation signal sequence that codes for a polyadenylation signal in pre-mRNA signaling proteins to cut it free 10-35 NT later. The strand needs further processing

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RNA Processing• During RNA Processing, both ends of the primary transcript are altered, interior sections are sometimes excised

– Alteration of ends:• 5’ end gets 5’ cap - modified guanine added after first 20-40 NT are transcribed• 3’ gets a poly-A tail - 50-250 adenine NT added to the 3’ end after the polyadenylation signal

– Both: -facilitate export of finished mRNA from the nucleus

-protect mRNA from degradation by hydrolytic enzymes

-help ribosomes attach to the 5’ end once mRNA reaches the cytoplasm• Untranslated regions (UTRs) do not code for proteins, have other functions like ribosome bonding, on both

the 5’ and 3’ ends

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–Introns may help evolution as increased spacing between exons increases the likelihood of crossing over during meiosis

–RNA Splicing = removal of large portions of an RNA molecule (primary transcript)

•Most eukaryotic genes have large non-coding regions between coding regions. Non-coding regions (introns) are cut out while coding regions (exons) are expressed•RNA Polymerase II transcribes both introns/exons, directed to splicing sites directed by small nuclear ribonucleoproteins (snRNPs) that recognize splice sites from the ends of introns

–snRNPs + proteins = spliceosome that cuts out introns, joins exons, catalyzes the process

•Ribozymes = RNA molecules functioning as enzymes. RNA can function as an enzyme because of: single strand, specifid 3-D structure, some bases have functional groups that can act as catalysts, can H-bond with other nucleic acids

•Some genes code multiple polypeptides depending on what sections of the primary transcript are treated as exons - alternative RNA splicing

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Translation• Transfer RNA (tRNA) transfers amino acids from the cytoplasm to the growing polypeptide chain in a ribosome

– tRNA tranlates mRNA using anticodons - complimetary sequences to the mRNA– Aminoacyl-tRNA syntetases bind 1 specific amino acid (20 types of syntetases)

to appropriate tRNA, tRNA with an amino acid attached = aminoacyl-tRNA (charged)• Some tRNAs can bond with multiple codons - wobble

– Ex. U at the end of 5’ on an anticodon can pair with A or G in 3’ codon. This is why most redundancies differ in the 3rd base of codons

• Ribosomes = made up of 3 sites: A (arrival), P (growing chain), E (exit). Large and small subunits– 1/3 proteins, 2/3 rRNA - most abundant form of RNA

• Initiation:– Small ribosomal subunit binds to mRNA with initiator tRNA (Met.) and various

initiation factors. Collectively make up translation initiation complex, large subunit then binds• Elongation:

– Amino acids added to c-terminus end of the preceding amino acid, divided into 3 steps:• Codon recognition - tRNA pairs with anticodon, GTP used for energy• Peptide bond formation - rRNA in large subunit catalyzes formation of peptide bonds

between amino group of new amino acid in the A site to the carboxyl end of the polypeptide in the P site

• Translocation - GTP used to move A siteP site, P siteE site– Multiple ribosomes can translate one mRNA at once - polyribosome

• Termination:– Elongation stops when stop codon reaches A site, release factor binds to the stop

codon in the A site, adds water molecule that cuts the polypeptide chain out of the P site

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After Translation• Proteins often fold on their own because of primary structure, sometimes aided by chaperonin• Amino acids may have sugars/lipids/phosphates added• Enzymes may remove amino acids form the amino (leading) end• Chain may be cut into multiple pieces• Signal polypeptides signal the ribosome making the polypeptide to either move to the rough ER or stay in the

cytoplasm

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Gene Expression• Prokaryotes regulate gene expression through regulating transcription

– Operons = operator (on/off switch)+promoter+genes controlled• Operons can be repressible (on unless turned off by a repressor protein) or inducible (off unless turned on

by an inducer)

• Eukaryotes don’t use operons– More condensed DNA sections (heterochromatin) less expressed– Histone acetylation promotes transcrption, methylation of bases (usually cytosine) does the opposite– Control elements = non-coding segments that serve as transcription factor binding sites

• Transcription factors = activators/repressors for enhancers and mediator proteins that help enhancers/promoters interact when far away

– Transcription can also be regulated through initiator proteins– Lifespan of mRNA also contributes to gene expression– Duration of gene’s expression can by regulated by Ubiquitin which binds to proteins to signal proteasomes to

degrade them

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Other Technologies• Gene cloning can be used to amplify a gene or produce a protein product ex. insulin

– Plasmids used as DNA vectors to get target genes into host cell. Restriction enzymes used to excise target genes, fragments = restriction fragments that are connected into the plasmid by DNA Ligase

• Polymerase Chain Reaction can quickly replicate specific DNA sequences in a test tube• Gel electrophoresis can determine fragment length, smaller ones go further in gel• Southern Blotting allows for detection of bands of a specific fragment in electrophoresed gel• First form of DNA sequencing = chain termination - looking at the last base added to determine overall sequence

– Now done as sequencing by synthesis - each base added is detected• Invitro mutagenesis = figuring out what certain genes do by turning them off

– RNA interference also finds gene function but by blocking translation or breaking down it’s mRNA

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Sources

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TextbookClass Notes

Images:

http://www.chemguide.co.uk/organicprops/aminoacids/dnachain2.gif

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