Replication, Transcription, and Translation. Corinne Landis Dale S. DiSalvo. The Basics—Eukaryotes vs. Prokaryotes. Eukaryotes . Prokaryotes . Unicellular (small in size) Lack a nucleus with a membrane—nucleoid area instead Lack organelles Replicate via budding or fission Ribosomes
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Replication, Transcription, and Translation Corinne Landis Dale S. DiSalvo
The Basics—Eukaryotes vs. Prokaryotes Eukaryotes Prokaryotes Unicellular (small in size) Lack a nucleus with a membrane—nucleoid area instead Lack organelles Replicate via budding or fission Ribosomes Cytoplasm Chromosomes to carry genes • Multicellular (larger in size) • Nucleus with membrane • Membrane enclosed organelles • Replicate via Mitosis or Meiosis • Ribosomes • Cytoplasm • Chromosomes to carry genes
Skeletal Structure IUPAC Name: (2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraen-1-ol (Retinol) Common Name: Vitamin A Formula: C20H30O
Biomolecule :Any molecule produced by a living organism • Carbs • Lipids • Proteins • Nucleic Acids
Central Dogma of Molecular Biology “The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.” -Francis Crick
Nucleotides • Building blocks for DNA and RNA
Structure of a Nucleotide • Five Carbon Sugar (Ribose or Deoxyribose) • 1+ Phosphate(s) (no phosphate=nucleoside) • 3’ end: Bears hydroxyl Group (Tail) • 5’ end: Bears phosphate group (Head)
Double Helical B-DNA • Three conformations of DNA found in nature • A, B, Z • B-DNA is prominent in cells • Conformation depends on • hydration level • DNA sequence • the amount and direction of supercoiling • chemical modifications of the bases • the type and concentration of metal ions • presence of polyamines in solution
DNA Replication Purpose: to replicate the entire DNA sequence in preparation for cell division—imperative for growth and repair
Key Enzymes in DNA Replication • Initiator Protein • Helicase • Primase • ssDNA binding proteins • DNA Polymerase • Clamp Protein • Topoisomerase • DNA Ligase • DNA Gyrase
Leading vs. Lagging Strand Leading: 3’ to 5’ direction Lagging: 5’ to 3’ direction
Editing Polymerase I • edits its own errors and those made by other enzymes • 3' --> 5' exonuclease removes incorporation errors using a second active site (editing or proofreading). Products: dNMPs • 5' --> 3' exonuclease activity resides on a separate domain; it removes nucleotides in lengths from 1 to 10 nucleotides.
DNA Replication: Prokaryotes vs. Eukaryotes • Main Difference: Prokaryotic chromosomes have a single origin of replication, while eukaryotic chromosomes have multiple origins of replication.Eukaryotes replicate at several points at the same time.
DNA Replication Video http://www.youtube.com/watch?v=teV62zrm2P0
Step by Step • Interphase • Prophase • Prometaphase • Metaphase • Anaphase • Telophase &Cytokinesis
Mitosis Video http://www.youtube.com/watch?v=VlN7K1-9QB0
Step by Step • The bacterium before binary fission is when the DNA tightly coiled. • The DNA of the bacterium has replicated. • The DNA is pulled to the separate poles of the bacterium as it increases size to prepare for splitting. • The growth of a new cell wall begins to separate the bacterium. • The new cell wall fully develops, resulting in the complete split of the bacterium. • The new daughter cells have tightly coiled DNA, ribosomes, and plasmids.
Binary Fission Video http://www.youtube.com/watch?v=DY9DNWcqxI4
Transcription • What is it? – Process by which RNA is synthesized using a DNA template • Why do it? – It lets cells control which genes they are using and when they are using them.
RNA Polymerase • Synthesizes RNA 5’-3’ • “Copies” the template DNA by complimentary base pairing rNTP’s • Able to unwind DNA • No need for single-strand binding protein • Activity is heavily regulated
Transcription: Initiation • Step 1 of the process • RNA polymerase binds to a promoter • TATA Box • Sequence specific promoter • DNA unwinds locally
Transcription: Elongation • rNTP’s enter RNA polymerase active site • rNTP’s are paired to bases of the DNA template • The growing transcript is polymerized 5’-3’ • DNA re-winds behind RNA polymerase
Transcription: Termination • How does RNA Polymerase know when to stop? • Terminator region of the DNA template • Many times, other proteins give RNA polymerase a cue to stop • Poly-A signal (eukaryotes) • Rho dependent termination (prokaryotes)
Special Considerations for Eukaryotes Initiation Processing mRNA gets a 5’ “Cap” and a Poly-A “Tail” Not everything that was transcribed should be translated Introns – “Interfere” Exons – “Expressed” snRNP’s remove the introns from the initial transcript • Upstream control elements • May be thousands of base pairs away • Activator Proteins • Transcription Factors • General • Gene Specific • Bind at the promoter
Eukaryotic Transcription http://www.youtube.com/watch?v=SMtWvDbfHLo
Special Considerations for Prokaryotes Initiation & Elongation Other No “Cap” or “Tail” for mRNA No introns Translation begins before transcription ends • Genes laid out in “operons” • Promoter • Operator – where regulatory proteins will bind • Structural Genes – several individual, but related genes within the same operon • Terminator • Inducible/Repressible
Prokaryotic Transcription http://www.youtube.com/watch?v=oBwtxdI1zvk
Translation • What is it? – the usage of the information encoded on mRNA to synthesize protein • Why do it? – Proteins are necessary for all cellular functions. They must be synthesized according to need. • Translaiton reads mRNA 5’-3’
The Ribosome • The cellular structure responsible for translation • Small and Large subunits • Reads information encoded in mRNA in triplet “Codons” • Obtains amino acids from charged tRNA • Synthesizes the encoded protein from N to C terminus
Codons • Groupings of 3 consecutive bases of mRNA • 1 codon encodes 1 amino acid • Some math tells us… • 3 bases/codon & 4 possible nucleotides/base… • 43 = 64 unique codons • But there are only 20 amino acids! • Each amino acid has multiple corresponding codons • We say the code is “redundant” • 3 codons are “Stop Codons” which tell the ribosome to stop translation