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Chapter 17 Protein Synthesis A Brief Review • 1) DNA Packaging; 2) DNA Replication: http://www.hippocampus.org/HippoCampus/ Biology;jsessionid=F30865A8DE688DE6D4 BD68F8161E8473 Overview of Transcription/Translation (in “real time”) • www.hippocampus.org is a great resource for almost every subject….not just Bio! • For this unit, specifically,Transcription and Translation: http://www.hippocampus.org/HippoCampu s/Biology;jsessionid=F30865A8DE688DE6 D4BD68F8161E8473 • Other recommended sites and videos: http://vcell.ndsu.nodak.edu/animations/ Introductory Questions #1 Name the substance that accumulates in a person’s urine causing alkaptonuria. 2) Why did Beadle and Tatum use breadmold spores to determine that one gene forms one polypeptide allowing for the first metabolic pathway to be defined? 3) Transcribe & Translate the following sequence of DNA by determining the nucleotide sequence for mRNA, the anticodon for tRNA, and the overall amino acid sequence: TACTCAGGACCTGCAACGATT mRNA: ??????????????????????????????? Amino acids Sequence: ??????????????????????????????? Anticodon: ??????????????????????????????? 1) 4) 5) How does the DNA and amino acid sequences differ from a person with sickle cell anemia and a person with normal hemoglobin in their RBC’s? (pg. 344) When mRNA is “processed” what is taken out (spliced)? Protein Synthesis: Chapter 17 Bridging the gap between Genotype & Phenotypes (proteins are thought to be that link) Trace the Flow of Information from Gene to Protein Key Topics: • • • • • • Garrod Beadle & Tatum Transcription (nucleus) Processing mRNA Translation (cytoplasm) Completed polypeptide (protein) Archibald Garrod (1909) • READ FROM TEXT: This is a synopsis • Main Point: First to suggest that genes dictate phenotypes through enzymes that catalyze specific chem rxns. This led to “one gene/ one enzyme” theory • Studied a rare genetic disorder: Alkaptonuria • Thought to be a recessive disorder • Tyrosine is not broken down properly into carbon dioxide and water. • An Intermediate substance: Alkapton (aka “Homogentisic acid”) accumulates in the urine turning it BLACK when exposed to air. • An enzyme was thought to be lacking • A genetic mutation was thought to be the cause “An Inborn Error of Metabolism” Garrod’s Conclusion • A mutation in a specific gene is associated with the absence of a specific enzyme. • Led to the idea of: “One Gene, One Enzyme” • Not validated until Beadle & Tatum’s work in the 1940’s with Neurospora (breadmold) George Beadle & EdwardTatum • Discovered the “One Gene, One Enzyme” Principle • Analyzed mutations that interfered with a known metabolic pathway • Organism they chose to work with: Neurospora (breadmold) -Grows easily -Grows as a haploid: (no homologs) -Mutants are easily identified: Dominant allele won’t be expressed • Neurospora can grow easily in only: salt, sugar, & Biotin (vitamin) George Beadle & EdwardTatum cont’d • Mutants-are unable to make certain organic molecules: amino acids, lipids, etc. • These substances are added to the media which will allow mutants to grow successfully • Exposed the haploid spores to x rays & UV to induce mutations • Haploid spores were crossed, grown in a variety of media to determine what kind of mutation was occurring • **They examined the effect of the mutation instead of identifying the enzyme. Beadle & Tatum Experiment w/Neurospora Beadle & Tatum’s Conclusion “One Gene affects One Enzyme” Later Revised “One Gene affects One Protein” Later Revised “One Gene affects One Polypeptide Chain” THE FLOW OF GENETIC INFORMATION (central dogma) DNA → RNA → PROTEIN • The information constituting an organism’s genotype is carried in its sequence of bases Protein Synthesis: overview • Transcription: synthesis of mRNA under the direction of DNA • Translation: actual synthesis of a polypeptide under the direction of mRNA Genetic Information Written in Codons is Translated into Amino acid Sequences • The “words” of the DNA “language” are triplets of bases called codons – The codons in a gene specify the amino acid sequence of a polypeptide – Why triplets? *Remember there are 20 amino acids and only 4 bases Various Types of RNA Molecules Gene 1 Gene 3 DNA molecule Gene 2 DNA strand TRANSCRIPTION RNA Codon TRANSLATION Polypeptide Figure 10.7 Amino acid 4) How many sites are present in the ribosome? Name the enzyme that is used to attach an amino acid to the tRNA molecule. • An exercise in translating the genetic code Transcribed strand DNA Transcription RNA Start codon Stop codon Translation Figure 10.8B Polypeptide The Genetic Code Dictionary • Virtually all organisms share the same genetic code • 1st codon determined was “UUU” by Marshal Nirenberg in 1961. • All of the codons were determined by the mid 1960’s Figure 10.8A RNA polymerase • In transcription, the DNA helix unzips – RNA nucleotides line up along one strand of the DNA following the base-pairing rules – The single-stranded messenger RNA peels away and the DNA strands rejoin DNA of gene Promoter DNA Initiation Elongation Terminator DNA Area shown in Figure 10.9A Termination Growing RNA Completed RNA Figure 10.9B RNA polymerase Transcription • Occurs in the nucleus • RNA Polymerase is needed -Adds nucleotides to the 3’ end only -Eukaryotes have three types vs. Bacteria with only one type • Elongation occurs from 5’ 3’ direction • TATA Box : initiation site for the attachment of RNA polymerase • 3 Steps: Initiation Elongation Termination Transcription: Initiation • RNA Polymerase binds to the “Promoter” region on the DNA (upstream about 25 nucleotides) • RNA Polymerase recognizes this region because of the “TATA” box • Other proteins also are needed: “Transcription factors” Transcription produces genetic messages in the form of RNA RNA polymerase RNA nucleotide Direction of transcription Template strand of DNA Figure 10.9A Newly made RNA Transcription: Elongation • DNA is untwisted (hydrogen bonds are broken) • About 10 base pairs are exposed • Nucleotides are are added to the 3’ end of the growing mRNA molecule • Proceeds at a rate of: ~ 60 nucleotides/sec Transcription: Termination • Termination site is “signaled” and “read” by RNA Polymerase • In Eukaryotes, a polyadenylation sequence (“AAUAAA”) is transcribed about 10-35 nucleotides before the (pre)mRNA is released • Pre-mRNA molecule is made consisting of “Coded” (Exons) and “Non-coded” (Introns) regions • Note: In Bacteria, Translation can occur as it is released from the first transcription event Stages of transcription • Transcription- The three Phases Eukaryotic RNA is processed before leaving the nucleus • Noncoding segments called introns are spliced out (they stay in the nucleus) • A cap and a tail are added to the ends • Exon Intron Exon Intron Exon DNA Cap RNA transcript with cap and tail Transcription Addition of cap and tail Introns removed Tail Exons spliced together mRNA Coding sequence http://highered.mcgrawhill.com/sites/0072437316/student_view0/c hapter15/animations.html# NUCLEUS CYTOPLASM Figure 10.10 mRNA Structure • 1) 5’ cap: modified guanine; protection; recognition site for ribosomes • 2) 3’ tail: poly(A) tail (adenine); protection; recognition; transport • 3) RNA splicing: involves introns & Exons • Exons (expressed sequences) retained (exit the nucleus to ribosome) • Introns (intervening sequences) -These are spliced out / spliceosome Key Regions on Newly Transcribed mRNA Splicesomes w/ SNRNP’s (Small nuclear Ribonuclearprotein-aka snurps) Animated View of Transcription • http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter15/animations.html# Translation • Occurs in the Cytoplasm • Key molecules and structures include: – – – – – mRNA tRNA Ribosome (30s and 40s subunits) Free floating amino acids Endoplasmic reticulum 3 Stages of translation • 1) Initiation – Brings together mRNA, tRNA and ribosome – Begins at the start code, AUG 2) Elongation -codon of mRNA and anticodon of tRNA complementary bond to one another -peptide bond fromation -translocation-ribosome moves to next codon 3)Termination-mRNA stop codons-UAA,UAG,UGA Translation: Overview Transfer RNA molecules serve as interpreters during translation • In the cytoplasm, a ribosome attaches to the mRNA and translates its message into a polypeptide • The process is aided by transfer RNAs Amino acid attachment site Hydrogen bond RNA polynucleotide chain Anticodon Figure 10.11A A Typical tRNA Molecule Translation: Transfer RNA (tRNA) mRNA from nucleus is ‘read’ along its codons by tRNA’s anticodons at the ribosome tRNA – has the anticodon and amino acid attached • Each tRNA molecule has a triplet anticodon on one end and an amino acid attachment site on the other Amino acid attachment site Anticodon Figure 10.11B, C Attachment of an Amino Acid Ribosomes Build Polypeptides Next amino acid to be added to polypeptide Growing polypeptide tRNA molecules P site A site Growing polypeptide Large subunit tRNA P A mRNA mRNA binding site Codons mRNA Small subunit Figure 10.12A-C • mRNA, a specific tRNA, and the ribosome subunits assemble during initiation Large ribosomal subunit Initiator tRNA P site A site Start codon mRNA 1 Figure 10.13B Small ribosomal subunit 2 Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation • The mRNA moves one codon at a time relative to the ribosome – A tRNA pairs with each codon, adding an amino acid to the growing polypeptide Amino acid Polypeptide A site P site Anticodon mRNA 1 Codon recognition mRNA movement Stop codon New peptide bond 3 Translocation 2 Peptide bond formation Figure 10.14 New peptide bond forming Growing polypeptide Codons Stage 4 Elongation A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time. mRNA Polypeptide Stop Codon Figure 10.15 (continued) Stage 5 Termination The ribosome recognizes a stop codon. The polypeptide is terminated and released. Translation- the Ribosome (2 subunits- large and small) rRNA site of mRNA codon & tRNA anticodon coupling P site holds the tRNA carrying the growing polypeptide chain A site holds the tRNA carrying the next amino acid to be added to the chain E site discharged tRNA’s Animated View of Transcription • http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter15/animations.html# Translation- Summary • Initiation ~ union of mRNA, tRNA, small ribosomal subunit; followed by large subunit • Elongation ~ •codon recognition; •peptide bond formation; •translocation • Termination ~ ‘stop’ codon reaches ‘A’ site • Polyribosomes: translation of mRNA by many ribosomes (many copies of a polypeptide very quickly) Transcription & Translation in Prokaryotes (*no nucleus) Introductory Questions 1) Transcribe & Translate the following sequence of DNA: TACTCAGGACCTGCAACGATT mRNA: ??????????????????????????????? Amino acids Sequence: Anticodon: 2) How does the DNA and amino acid sequences differ from a person with sickle cell anemia and a person with normal hemoglobin in their RBC’s? 3) When mRNA is “processed” what is taken out (spliced)? 4) How many sites are present in the ribosome? Name the enzyme that is used to attach an amino acid to the tRNA molecule. Mutations & Errors During Transcription & Translation DNA Repair • Mismatch repair: DNA polymerase • Excision repair: Nuclease • Telomere ends: telomerase Mutations can change the meaning of genes • Mutations are changes in the DNA base sequence – These are caused by errors in DNA replication or by mutagens – The change of a single DNA nucleotide causes sickle-cell disease Mutation: Some Definitions • • • • A heritable change in the genetic material Mutations may be neutral, beneficial, or harmful Mutagen: Agent that causes mutations Spontaneous mutations: Occur in the absence of a mutagen Protein Synthesis and Mutation • Mutation(Permanent, heritable DNA changes) – Point mutation (base substitutions) • Missense mutation • Nonsense mutation (premature stop) • Silent mutation – Insertions/deletions • Frameshift mutation – Dramatic change in amino acids – Run-ons, premature stops (nonsense mut.) • The Creation of Mutation (mutagenesis) – Spontaneous mutation • Occurs in DNA replication (1 in 109 bp) – Chemical mutagens • Increases rate to 1 in 1000-100,000) – Radiation • X rays, gamma rays break DNA, bases • UV light causes knots in DNA strand • Types of mutations NORMAL GENE mRNA Protein Met Lys Phe Gly Ala Lys Phe Ser Ala BASE SUBSTITUTION Met Missing BASE DELETION Met Lys Leu Ala His Figure 10.16B Normal hemoglobin DNA mRNA Mutant hemoglobin DNA mRNA Normal hemoglobin Sickle-cell hemoglobin Glu Val Figure 10.16A Mutation: Base Substitution (Point Mutations) G G C C Glu (a) Silent mutation (d) Run-on mutation Frameshift Mutations: Insertions/Deletions THEBIGCATATETHERAT THEBIGCBATATETHERAT Figure 8.17a, d Summary: Mutations POINT mutations-one base pair is changed (*may be more than one in same gene). Most common. – Substitutions (silent, missense, nonsense) FRAMESHIFT: Alters reading frame – Insertions (additions) – Deletions • Mutagens-physical and chemical agents that interact DNA to cause mutations—exp.: xrays Mutations: genetic material changes in a cell