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 (breaks Hydrogen bonds), creating a replication fork (binds AT 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 (prevents Hydrogen bonds)—binds after replication fork

• Topoisomerase- Reduces/relieves strain caused by unwinding ahead of replication forks by breaking, swiveling, and rejoining parental DNA (breaks COVALANT bonds in DNA backbone)

• 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 for Primer (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

  • *From the perspective of the DNA strand moves from the 3’ end towards the 5’ end;

  • Adds bases to the 3’ end of the PRIMER

    • 

  • Replication of leading strands moves TOWARDS the replication forks; Replication of lagging strand (Okazaki fragments) moves AWAY from replication forks

    • Order: Both leading strands synthesized first then each segment of lagging strand closest to the leading strand

      • 

• DNA polymerase I- Replaces RNA primers (at the ends) 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 (modified guanine nucleotide) and a 3’ poly-A tail (long string of adenine nucleotides) 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 (ribosomes are one big ribozyme)

  • 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)

  • Anti-codon Calculations:

    • Anti codons are ANTIPARALLEL to the codon in the 3’ to 5’ direct → reverse pairing of codons

• 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

    • 

3 Steps of Translation (similar to transcription)

• 1. Initiation

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

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

  • Large ribosomal subunit attaches to the complex forming the Translation Initiation Complex

  • 

• 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 (process of sliding down one codon on the mRNA)

    • 

• 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

• Phosphorylation

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- amino acid replaced by 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

    • *The insertion or deletion of three nucleotide pairs does not cause a frameshift mutation; Insertion/deletion of one nucleotide causes frameshift

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

Directionality:

DNA replication:

• Leading 5’ → 3’; Lagging 3’ → 5’;

• New DNA strands synthesized from 5’ → 3’ direction

RNA transcription

• Template strand 3’ → 5’

• Coding strand 5’ → 3’

• mRNA 5’ → 3’

RNA translation

• mRNA 5’ → 3’

• tRNA anticodons 3’ → 5’

Practice Problems:

• The -OH group on the 3' carbon of the sugar unit is the attachment site for the nitrogenous base. False

• Complementary base pairing relies on the number of hydrogen bonds that each base can make. True

• In a single nucleotide, the phosphate group is attached to the 5' carbon of the sugar unit. True

• The antiparallel arrangement of double-stranded DNA is due to the phosphate group being bonded to the 3' carbon on one strand and the 5' carbon on the complementary strand. False

• The phosphate attached to the 5' carbon of a given nucleotide links to the 3' -OH of the adjacent nucleotide. True

FRQ

Gibberellin is the primary plant hormone that promotes stem elongation. GA 3-beta-hydroxylase (GA3H) is the enzyme that catalyzes the reaction that converts a precursor of gibberellin to the active form of gibberellin. A mutation in the GA3H gene results in a short plant phenotype. When a pure-breeding tall plant is crossed with a pure-breeding short plant, all offspring in the F1 generation are tall. When the F1 plants are crossed with each other, 75 percent of the plants in the F2 generation are tall and 25 percent of the plants are short.

The wild-type allele encodes a GA3H enzyme with alanine (Ala), a nonpolar amino acid, at position 229. The mutant allele encodes a GA3H enzyme with a threonine (Thr), a polar amino acid, at position 229. Describe the effect of the mutation on the enzyme and provide reasoning to support how this mutation results in a short plant phenotype in homozygous recessive plants. (2 points)

This mutation that alters the amino acid from polar to nonpolar would alter the structure specifically the tertiary structure and thus the function of the protein thus could decrease or stop the production of gibbrellin resulting in short plants

wild type gene is dominant so in heterozygous the gene would be expressed still expressing prodcution of