Today: Reviewing Mitosis/ Exploring the Function of Taxol Structure and Function of DNA!

Key Experiments in the Discovery of DNA: Griffith’s Transformation Experiment (1928) What do we learn about the nature of the genetic material from this work??

Hershey and Chase Key Experiments in the Discovery of DNA: Hershey and Chase study Bacteriophage Bacteriophage inject their genetic material into host cells to replicate themselves. If Hershey and Chase can figure out what is injected, they can identify the “genetic material”!

The Hershey-Chase Experiment…

The accumulation of evidence: Prior to mitosis (cell division) a eukaryotic cell doubles its DNA content, distributing the DNA equally between the two daughter cells. Somatic cells have twice as much DNA (diploid) as gametes (haploid) Chargaff: DNA composition varies from one species another and in the DNA of any one species the amounts of the four bases are not equal, but do occur in a predictable ratio: (A=T, C=G)

You Try: What are the key features of this model? Watson and Crick You Try: What are the key features of this model? With help from others, Watson and Crick decipher the structure of DNA! (1953)

PURINES PYRIMIDINES Pairing of PURINES with PYRIMIDINES is key to both Structure and Replication of DNA

Reviewing the Mechanics of DNA Replication: Must be FAST and ACCURATE (~1 mistake/billion bases)!

DNA Replication: A more Complete View

Another Challenge: The End Replication Problem As eukaryotes replicate their DNA, they are unable to “fill-in” the 5’ end of new molecules.

The Solution: Telomeres- noncoding multiple repeats of a short nucelotide sequence (TTAGGG) Protects genes (coding sequences) from being eroded. (Erosion triggers programmed cell death!) Would you expect telomerases to be active in all cells? If not, where would you expect to find active telomerases??

Next Up: The Missing Link!

Getting From a Gene to a Polypeptide Occurs in 2 Steps: TRANSCRIPTION: transcribing DNA into RNA 2. TRANSLATION: translating RNA into an amino acid sequence ?

Prokaryotic Transcription & Translation In Prokaryotes, transcription and translation can occur simultaneously.

Eukaryotic Transcription & Translation In Eukaryotes, transcription and translation are separated. Transcription occurs in the nucleus, generates a primary transcript (pre mRNA) which is edited and exported as mRNA.

In order to translate DNA (RNA) you must first crack the genetic “code”! The genetic code is a triplet code in which a group of three bases (codon) of a DNA molecule code for a particular amino acid.

Reading the Genetic Code: Note that the code is sometimes redundant, but never ambiguous. It is also (nearly) universal, allowing for DNA from one species to function in another! (transgenic organisms)

Arabidopsis thaliana C24 wild type (left) and transformed (GFP; right) Tobacco plant expressing a firefly gene (luciferase) Herman, at right, is the first transgenic dairy animal engineered to make the human milk protein, lactoferrin, which is an antibacterial protein that can be used to treat immunosuppressed patients and could be incorporated into infant formula.

TRANSCRIPTION occurs in 3 Steps: Initiation Elongation, and Termination

Initiation of Transcription: Important Details!

Step 2: Elongation Once the RNA polymerase is attached to the promoter DNA, the DNA strands unwind, and the RNA Polymerase begins to TRANSCRIBE the template strand.

Step 3: Termination Transcription continues until AFTER the RNA polymerase transcribes a TERMINATOR SEQUENCE. This transcribed terminator (RNA) acts as the signal!

Step 3: Termination In prokaryotes, transcription generally stops right at the end of the termination signal, with the polymerase releasing both the RNA and DNA molecules.

Step 3: Termination In eukaryotes, the polymerase may continue for thousands of bases past the termination signal (AAUAAA). ~10-15 bases past this termination signal the pre-mRNA is cut free from the enzyme for RNA PROCESSING Let’s Watch! http://www.dnai.org/lesson/go/19436/15510

RNA Processing: 1. Modifying the ends of the pre-mRNA 2. Splicing the interior of the pre-mRNA At the 3’ end, and enzyme attaches and creates a poly(A) tail (50-250+ adenine nucleotides). This 3’ poly(A) sequence: 1. inhibits degradation 2. assists with ribosome binding 3. facilitates the export of the mRNA from the nucleus. The 5’ end is immediately “capped” with a modified guanine (G). This 5’ cap: 1. protects the molecule from degradation 2. is recognized by the ribosome to initiate translation

ASIDE: Understanding RNA processing provides enormous advantages for applying Genetic Technology! Example: RT-PCR Extract RNA, not DNA

RNA Processing: Step 2. Splicing the interior of the pre-mRNA Eukaryotic genes contain INTRONS (long noncoding regions of nucleotides that are not translated). An average transcription unit is ~8000 nucleotides, but only ~1200 nucleotides are used to code for an average protein. (**How many amino acids in an average protein?)

The Mechanism for removing INTRONS Short nucleotide sequences at the ends of introns act as signals for splicing. Small nuclear ribonucleoproteins (snRNPs) recognize these sites. Several snRNPS combine with additional proteins to form a SPLICEOSOME. The SPLICEOSOME cuts at specific points, and joins the ends of the two exons together.

Why Introns??

Possible Functions/Benefits of RNA Splicing: Regulatory Role? (Transcriptional regulation of gene expression) Alternative RNA splicing (make many different proteins from the same “gene”) Facilitate the evolution of new proteins (DOMAINS may be exchanged during crossover. Introns provide more places for crossover to occur.)

From mRNA to Protein Edited mRNA is then exported from the nucleus to the cytoplasm where the protein will be assembled

Translation: From mRNA to Protein mRNA is translated by an interpreter, transfer RNA (tRNA). tRNA transfers the correct amino acid from the cytoplasm to a ribosome. The ribosome adds amino acids to the growing end of a polypeptide chain.

tRNA: Each type of tRNA molecule carries a specific amino acid, and contains a specific anticodon. The anticodon is a nucleotide triplet that binds with its complementary codon on mRNA

tRNA’s: Note the directional nature of this process! mRNA’s are typically read 5’  3’. Therefore anticodons are 3’ 5’.

For this system to work, the correct amino acid must be attached only to tRNA molecules carrying the correct anticodon. Correct attachment is catalyzed by one of 20 different Aminoactyl-tRNA synthetase enzymes. Endergonic? Exergonic?

Ribosome Structure: Ribosomes are made of two subunits (the large and small). Each subunit is made of proteins and ribosomal RNA (rRNA). Eukaryotes assemble their ribosomes in the nucleus (importing the protein component from the cytoplasm). The subunits are then exported to the cytoplasm, where they assemble upon binding a mRNA molecule. Mouse liver

(ex. Tetracycline, streptomycin) Differences in Eukaryotic and Prokaryotic Ribosome Structure Make Good Drug Targets! (ex. Tetracycline, streptomycin)

3 binding sites for tRNA: P site- holds the tRNA carrying the growing polypeptide chain A site- holds the tRNA carrying the next amino acid 3. E site- where the discharged tRNAs leave the ribosome. (The ribosome is an ENZYME that catalyzes the formation of a peptide bond between amino acids!)

TRANSLATION also occurs in 3 steps: Initiation- the RIBOSOME assembles on the mRNA molecule at the START CODON Elongation- the polypeptide chain is assembled sequentially as amino acids are covalently bound to one another Termination- the Ribosome reaches a STOP CODON and releases the mRNA and Polypeptide

Initiation- The small ribosomal subunit binds to a mRNA An initiator tRNA (UAC) pairs with the start codon (AUG) The large ribosomal subunit binds to complete the Translation Initiation Complex. Initiation, Elongation, and Termination Initiation requires energy and initiation factors

Step 2: Elongation Elongation Factor Requires energy! mRNA moves through the ribosome

Amino Acid Structure and the Formation of Polypeptides A quick review: Amino Acid Structure and the Formation of Polypeptides Note the Amino and Carboxyl ends. (Also called the N-terminus and the C-terminus)

Step 3: Termination Let’s Try! Elongation continues until a stop codon in the mRNA reaches site A. A RELEASE FACTOR protein binds to the stop codon in the A site, causing the completed polypeptide to be released from the ribosome. Let’s Try!

Many single ribosomes can translate a given mRNA molecule simultaneously (POLYRIBOSOMES).

Post-Translational Modification Sugars, lipids, phosphate groups, etc. may be added to amino acids in the polypeptide Amino acids may be cleaved (removed) from the polypeptide, or the entire chain may be cut into multiple pieces

Getting Proteins Where They’re Going: Signal Peptides All ribosomes begin assembling proteins in the cytoplasm (free ribosomes). If a signal peptide is present, they bind to the ER (bound ribosomes)

Getting Proteins Where They’re Going: Signal Peptides

Understanding Transcription and Translation Allows You to Predict the Effect of Mutations!

Understanding Gene Mutations 1. Base-pair SUBSTITUTIONS- What happens if UCA UCC ?

Understanding Gene Mutations 1. Base-pair SUBSTITUTIONS- What happens if UAC UAG?

Sickle Cell Disease: a Substitution

The Effects:

Understanding Gene Mutations 2. INSERTIONS and DELETIONS One+ base pairs are inserted or deleted from a DNA sequence. Insertions and Deletions frequently result in FRAMESHIFT MUTATIONS. Example: CCR5 and HIV/Plague Resistance!

Insertions or Deletions Can Result in Missense or Nonsense