AP Bio Unit 6

Evidence that DNA is the Genetic Material (Scientific Contributions to DNA)

Frederick Griffith

• Transformation principle—change in genotype and phenotype due to assimilation of external DNA by a cell

• Experiment showed that living R bacteria transformed into deadly S bacteria by unknown heritable substance

Avery, McCarty, MacLeod

• Discovered the transforming agent was DNA

• Tested DNA, RNA and proteins in heat-killed pathogenic bacteria, only when DNA was destroyed did transformation not occur

Hershey and Chase

• Proved DNA is the genetic material

• Used bacteriophages found that when bacteriophage DNA entered hosts it affected the cells but the proteins did not

Edwin Chargaff

• *Chargaff’s Rules

  • Ratios of Adenosine = Thymine and Guanine = Cytosine are THE SAME (pairs)

  • DNA composition varies between species

Rosalind Franklin

• Provided images of the structure of DNA using X-ray crystallography

• Provided measurements on chemistry of DNA

James Watson and Francis Crick

• Discovered the double helix structure of DNA by building models of DNA confirming Franklin’s X-ray data and Chargaff’s rules

• Discovered semiconservative model of DNA replication

DNA Structure

• Double helix sugar (deoxyribose) + phosphate backbone

  • Phosphodiester bonds- bonds connecting phosphate group to sugar (Deoxyribose—DNA; Ribose—RNA)

• Nitrogenous bases rungs

  • Adenine—Thymine (2 hydrogen bonds)

  • Guanine—Cytosine (3 hydrogen bonds)

  • Adenine + Guanine are purines (larger); Thymine and Cytosine are pyrimidines (smaller)

    

• Antiparallel strands

  • One strand (leading) runs from 5’ → 3’ (phosphate group on top)

  • Other strand (lagging) runs upside-down in the opposite 3’ → 5’ direction (phosphate group on the bottom)

    

How DNA is packaged

• DNA starts as a double-stranded molecule

• DNA coils around histone proteins to form nucleosomes (like beads on a string)

• Nucleosomes coil into chromatin structures

• Chromatin further condensed into chromosomes during cell division

  

DNA Replication

• Semiconservative model- Two strands of parental DNA separate with each serving as a template for synthesis of a new complementary strand

  

Steps of DNA Replication:

• Helicase- unwinds DNA at the origins of replication (stretches of DNA with specific sequences), creating a replication fork

• Initiation proteins attach to DNA to separate them, creating a replication bubble

• Single-strand binding proteins (SSBs) bind to unpaired DNA strands to prevent strands from re-pairing

• Topoisomerase- Reduces/relieves strain caused by unwinding ahead of replication forks by breaking, swiveling, and rejoining parental DNA

• Primase synthesizes RNA primers (5-10 nucleotides long) to provide a starting point for replication of the new DNA strand (starts at 3’ end of RNA primer)

  

• DNA polymerase III- Synthesize new DNA by adding complimentary bases to with the new DNA made in 5’ → 3’ direction (new bases added to the 3’ end)

  • Adds bases to the lagging strand with the addition of Okazaki fragments- short sections of DNA formed due to discontinuous synthesis of the lagging strand

  • Replication of leading strands moves TOWARDS the replication forks; Replication of lagging strand moves AWAY from replication forks

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• DNA polymerase I- Replaces RNA primers with DNA nucleotides

• DNA ligase- joins all Okazaki fragments into a continuous strand

• Proofreading and Repair

  • DNA polymerases proofread each nucleotide against template, removing any incorrect nucleotides

  • Mismatch repair- other enzymes remove and replace incorrectly paired nucleotides from replication errors (evaded proofreading)

  • Nucleotide Excision Repair- Nucleases cut damaged DNA; DNA polymerase + ligase fill in missing nucleotides/gaps

• Telomeres

  • Since DNA polymerase only adds nucleotides to the 3’ ends, no way to complete 5’ ends of the daughter strands → DNA strands grow shorter and shorter over many replications

  • Telomeres: Repeated units of short nucleotide sequences (TTAGGG) at the ENDS of DNA serve to “cap” the ends of DNA to postpone erosion of genes at the ends

  • Telomerase: Enzyme that lengthens/extends telomeres in eukaryotic germ cells to restore to their original length (not active in most somatic cells shows inappropriate activity in some cancer cells)

Gene Expression

Gene Expression- process by which DNA directs the synthesis of proteins/RNA

• One gene-one RNA molecule (which can be translated into a polypeptide)

Flow of Genetic Information

• Central dogma: DNA → RNA → Protein

  • Transcription- Process of transcribing DNA sequence into RNA (DNA → RNA)

  • Translation: Translation sequence of mRNA into amino acids during protein synthesis (RNA → proteins)

Roles/Types of RNA

• mRNA- carries code from DNA that specifies amino acids

• tRNA- carries specific amino acid to ribosome based on its anticodon to mRNA codon

• pre-mRNA- precursor to mRNA newly transcribed and not edited

• rRNA- makes up 60% of the ribosome and the site of protein synthesis

• ribozyme- RNA that functions enzymes

• RNAi- interference RNA a regulatory molecule

• srpRNA- signal recognition particle that binds to signal peptides

• snRNA- small nuclear RNA part of a spliceosome has structural and catalytic roles

• miRNA/siRNA- micro/small interfering RNA; binds to mRNA or DNA to block it for regulating gene expression/cutting it up

Genetic Code

• One DNA strand (3’ → 5’) serves as the template strand

• mRNA is complimentary to template (5’ → 3’)

• mRNA codons/triplets code for amino acids (read in groups of 3)

• ALWAYS starts with the start codon (AUG) and ends with 3 possible end codons (UAA, UAG, UGA)

Transcription steps

• Transcription unit: Stretch of DNA that codes for a polypeptide or RNA (i.e. tRNA, rRNA)

• RNA Polymerase

  • Separates DNA strands and transcribes mRNA

  • mRNA elongates in 5’ → 3’ direction

  • Attaches to promoter (start of gene) and stops at the terminator (end of gene)

• 1. Initiation-

  • After RNA polymerase BINDS to the promoter, polymerase unwinds the DNA strands and initiates RNA synthesis at the start point of the template strand

    • Promoter- DNA sequence where RNA polymerase attaches and initiates transcription; in bacteria, the sequence signaling the end of transcription is the terminator

    • Transcription unit- the stretch of DNA downstream from the promoter that is transcribed into an RNA molecule

  • In Prokaryotes:

    • Bacteria: RNA polymerase binds DIRECTLY to promoter in DNA

  • Eukaryotes:

    • TATA box- DNA sequence (TATAAAA) in the promoter region upstream from transcription start site

    • Transcription factor recognize the TATA box before RNA polymerase binds to the DNA promoter

    • Transcription factors + RNA polymerase = transcription initiation complex

      • 

• 2. Elongation

  • RNA polymerase moves downstream, unwinding DNA and elongating the RNA transcript by adding RNA nucleotides to the 3’ end of the growing chain

  • After mRNA is made, DNA strands are retwisted to reform a helix

• 3. Termination

  • RNA polymerase transcribes a terminator sequence (prokaryotes) OR polyadenylation signal sequence (eukaryotes) then mRNA and polymerase detach

    • Becomes pre-mRNA for eukaryotes, and ready-to-use mRNA for prokaryotes

  • RNA transcription is released and polymerase detaches from DNA

RNA processing (Eukaryotes)

• Alternations to pre-mRNA ends for eukaryotes after transcription

  • 5’ cap and a 3’ poly-A tail are added to pre-mRNA

    

    • Functions:

      1. Export from nucleus

      2. protect mRNA from enzyme degradation

      3. Attach mRNA to ribosomes

• RNA Splicing:

  • Introns (noncoding sequences) are CUT OUT while exons (code for amino acids) are joined together

    • Small nuclear ribonucleoproteins (snRNPs) are made of snRNA + protein

    • snRNPs recognize splice sites and join with other proteins to form a spliceosome—catalyze the process of removing intros/joining exons

    • Ribozyme- RNA acts as enzyme

  • Introns-

    • Serve to regulate gene activity

    • Alternative RNA Splicing- produce different combinations of exons

Translation

• Takes place inside ribosomes

• tRNA (transfer RNA)

  • Transcribed in the nucleus

  • Specific to each amino acid and transfers amino acids to ribosomes based on Anticodons- pairs with complementary mRNA codon

  • Wobble- base-pairing rules between THIRD base of codon and anticodon not as strict (different nucleotide for third codon can code same amino acid)

• Aminoacyl-tRNA-synthetase- enzyme that binds tRNA to specific amino acid

• Ribosomes-

  • rRNA + proteins; Made in the nucleol; Comprised of 2 subunits

  • Actives Sites

    • A site: Holds the Amino Acid (AA) to be added

    • P site- Holds growing polypeptide chain

    • E site- Exit site for tRNA

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3 Steps of Translation (similar to transcription)

• 1. Initiation

  • Small subunit binds to the start codon (AUG) on mRNA

  • tRNA carrying met (start amino acid) attaches to P site

  • Large subunit attaches

  • 

• 2. Elongation

  • Codon recognition- tRNA anticodon matches codon in A site

  • Peptide bond formation- Amino acids in A site bonds with peptide in P site

  • Translocation- tRNA in A site moves to P site while tRNA in P site moves to E site to exit

    • 

• 3. Termination

  • Stop codon reached to stop translation

  • Release factor binds to stop codon and polypeptide released

  • Ribosomal subunits dissociate

    • 

Protein Folding

• During synthesis, polypeptide chain coils and folds spontaneously

• Chaperonin: protein that helps polypeptide fold CORRECTLY

Post-Translational Modifications

• Attaches sugars, lipids, phosphate groups, etc.

• Remove amino acids from ends

• Cut into several pieces

• Subunits come together

Types of Ribosomes:

• Free ribosomes: synthesize proteins that stay in cytosol and function there

• Bound ribosomes (attached to ER) make proteins for secretion and proteins of the endomembrane system (nuclear envelope, ER, Golgi, lysosomes, vacuoles, plasma membrane)

  • Uses signal peptide- 20 amino acids at the leading end of polypeptide to determine location

  • Signal-recognition particle (SRP) brings the ribosome to the ER

• Polyribosomes- a single mRNA can be translated by several ribosomes at the same time

  • Prokaryotes can transcribe + translate at the SAME TIME

Mutations:

• Changes in the genetic material of a cell

• Chromosomal mutations- large-scale, causes disorders or death

  • i.e. Nondisjunction (extra chromosomes), translocation, inversion, duplication, large deletions

• Point mutations- alter a single nucleotide pair of a gene

  • Substitution- replacing one with another

    • Silent- same amino acid (often when last codon is altered but still codes for same amino acid)

    • Missense- different amino acid

    • Nonsense- stop codon instead of amino acid

    • *Sickle cell disease is caused by a point mutation resulting in the VAL mutant protein instead of GLU

  • Frameshift (insertion/deletion)- mRNA read incorrectly leads to nonfunctional proteins

• Mutagens- substances of forces that cause mutations in DNA (radiation, chemicals, infectious agents)