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5 DNA RNA Protein Synthesis
 

5 DNA RNA Protein Synthesis

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    5 DNA RNA Protein Synthesis 5 DNA RNA Protein Synthesis Presentation Transcript

    • DNA, RNA, and Protein Synthesis
    • Importance and Structure of DNA: Deoxyribo-Nucleic Acid
      • Historical Review:
        • 1900’s – Morgan’s studies with fruit flies showed that genes were located on chromosomes and chromosomes consisted of protein and DNA
        • 1952- Hershey-Chase demonstrated that DNA (not protein) was the genetic material of a viral phage
    • Figure 16.2a The Hershey-Chase experiment: phages
    • Figure 16.2b The Hershey-Chase experiment
    • Phages Infecting a bacterium
    • Figure 16.1 Transformation of bacteria – Griffith (and later Avery, McCarty and MacLeod)
    • The Structure of DNA
      • Nucleotide monomers:
        • Phosphate
        • Pentose Sugar (C5) – Deoxyribose Sugar
        • Organic Nitrogen Base :
          • Cytosine (C)
          • Adenine (A)
          • Guanine (G)
          • Thymine (T)
    • Structure of DNA cont’
      • Polynucleotide chain with linkage via phosphates to next sugar, with nitrogen base away from backbone of
      • Phos-sugar-phos-sugar
      • Dehydration synthesis
    • Beginning of the 1950’s several labs were studying the structure of DNA
      • Maurice Wilkins & Rosalind Franklin
        • X-ray crystallography: x-rays pass through pure DNA and diffraction of x-rays were then examined on film
      • James Watson and Francis Crick did not have the expertise of Franklin and were without proper photos until………
    • Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA
    • Watson and Crick
    • April 1953 – Classical one page paper in Nature by Watson and Crick
      • A double helix – 2 polynucleotide strands
      • Sugar-phosphate chains of each strand are like the side ropes of a rope ladder
      • Pairs of nitrogen bases, one from each strand, form the rungs or steps
      • The ladder forms a twist every 10 bases (all from x-ray studies!)
    • Figure 16.5 The double helix
    • Internal Structure of DNA: Purine and pyridimine? REMEMBER X-RAY DATA
    • Confirms Erwin Chargaff’s Rules
      • # of Adenine = to # of thymine
      • # of guanine equal to # of cytosine
      • This dictates the combinations of N-bases that form steps/rungs
      • Does not restrict the sequence of bases along each DNA strand
    • Information storage in DNA
      • The 4 nitrogenous bases are the “alphabet” or code for all the traits the organism possesses
      • Different genes or traits vary the sequence and length of the bases
      • ATTTCGGAC vs. GGGATTCTAG vs. GATC
    • Replication/Duplication of DNA
      • Due to complimentary base paring – one strand of DNA determines the sequence of the other strand
      • Therefore, each strand of double stranded DNA acts as a template
      • The double helix first unwinds – controlled by enzymes –and uses new nucleotides that are free in the nucleus to copy a complimentary strand off the original DNA strand
    • Figure 16.7 A model for DNA replication: the basic concept (Layer 1)
    • Figure 16.7 A model for DNA replication: the basic concept (Layer 2)
    • Figure 16.7 A model for DNA replication: the basic concept (Layer 3)
    • Figure 16.7 A model for DNA replication: the basic concept (Layer 4)
    • Figure 16.8 Three alternative models of DNA replication
    • Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 1)
    • Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 2)
    • Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 3)
    • Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)
    • There are a series of enzymes that control DNA replication – enzymes which:
      • Uncoil the original double helix strand via a helicase
      • Single-strand binding protein keeps helix apart so replication can start
      • Prime an area to start replication – primase except it adds RNA nucleotides at first
      • Polymerase to join individual nucleotides (dehydration synthesis)
      • Ligases to join short segments
    • DNA REPLICATION
    • Figure 16.10 Origins of replication in eukaryotes
    • Figure 16.11 Incorporation of a nucleotide into a DNA strand
      • The carbons of the deoxyribose sugar are numbered
      • #3 carbon attached to an -OH group
      • #5 carbon holds the phosphate molecule of that nucleotide
      • #3 ready to bond with another nucleotide to form a polynucleotide link (5’ to3’)
      • Notice complimentary strand in opposite direction (5’ to 3’)
      • DNA always grows 5’ to 3’ never 3’ to 5’
      Antiparallel Arrangement of Double Strands
    • Definitions
      • Origins of Replication – where replication of the DNA molecule begins
        • Bacteria – circular DNA – 1 origin of replication (RF)
        • Eukaryotes – multiple origins of replication (ORFS)
          • ORF = Replication Fork
    • More Definitions
      • DNA Polymerases – enzymes that catalyze DNA replication
      • Leading Strand – Synthesized continuously towards the replication fork by the DNA polymerase in one long fashion
      • Lagging Strand – Synthesized by short fragments away from the replication fork by the DNA polymerase
    • Definitions Cont’
      • Ligase – combines (joins) short fragments
      • Primer – starts replication of DNA (in this case it’s RNA)
      • Primase – an enzyme that joins the RNA nucleotides to make the primer
      • Helicase – an enzyme that untwists the double helix at the replication fork
      • Nuclease – a DNA cutting enzyme
    • DNA REPLICATION -VIDEO
    • Figure 16.13 Synthesis of leading and lagging strands during DNA replication
    • Figure 16.14 Priming DNA synthesis with RNA
    • Figure 16.15 The main proteins of DNA replication and their functions
    • Figure 16.16 A summary of DNA replication
    • Figure 16.17 Nucleotide excision repair of DNA damage
    • A PROBLEM!
      • The end of the leading strand was initiated with an RNA primer
        • Normally removed by other DNA polymerase
      • Removal of gaps by DNA Polymerase doesn’t work on lagging strand end
        • RNA primer removed with no replacement
        • A GAP!
        • SHORTER AND SHORTER FRAGMENTS?
    • Prokaryotes have circular DNA – no problem at ends (there aren’t ANY!
      • Eukaryotes – have special terminal sequences of 6 nucleotides that repeat from 100-1000 times with no genes included
        • Telomers
      • Protect more internal gene materials from being eroded
      • Germ cells / sex cells have a special enzyme (telomerase) that actually restore shortened Telomers
      • Somatic cells – telomer continues to shorten and may play a role in aged cell death
      • Cancer cells
        • A telomerase prevents very short lengths
    • Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes
    • Ribonucleic Acid (RNA)
      • Structure of RNA
        • Nucleotide monomer
          • Phosphate
          • Pentose sugar = ribose (extra oxygen)
          • Nitrogenous base (A/G/C/U)
          • Single stranded
          • 3 types (mRNA, tRNA, rRNA)
    • Synthesis of RNA - transcription
      • DNA acts as a template, but only one strand of DNA utilized at a given time
      • This exposed strand is controlled by specific enzymes that pair the DNA nucleotides with free RNA nucleotides which are also present in the nucleus
      • These RNA nucleotides form a single stranded RNA nucleic acid
      • DNA = ATTGGCT
      • RNA = UAACCGA
      • Short segments of DNA are transcribed at a time with start and stop messages
    • Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 1)
    • Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 2)
    • Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 3)
    • Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 4)
    • Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 5)
    • Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 1)
    • Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 2)
    • Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 3)
    • Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 4)
    • Figure 17.6 The stages of transcription: elongation
    • Three types of RNA
      • mRNA : messenger RNA
        • Transcribed from a specific segment of DNA which represents a specific gene or genetic unit
      • tRNA : transfer RNA
        • Transcribed from different segments of DNA and their function is to find a specific amino acid in the cytoplasm and bring it to the mRNA
      • rRNA : ribosomal RNA
        • Transcribed at the nucleolus - with proteins function as the site of protein synthesis
    • Three types of RNA
    • Protein Synthesis = Translation
      • Ribosomes = sites of protein synthesis
        • 30% - 40% protein
        • 60% - 70% RNA (rRNA)
        • Assembled in nucleus and exported via nuclear pores
        • Antibiotics can paralyze bacterial ribosomes, but not eukaryotic ribosomes (not targeting them)
        • 2 ribosomal subunits –a large and a small
        • Small subunit has been used as a means of classifying different bacteria and different invertebrates (16S)
          • Eukaryotes – 18S
        • There are three sites on the ribosome that are involved in protein synthesis
    • Ribosomes bring mRNA together with amino acid bearing tRNA’s
      • Three ribosomal sites
        • P Site (peptidyl-tRNA) holds the tRNA carrying the growing peptide chain after several amino acids have been added
        • A site (aminoacyl-tRNA) holds the next single amino acid to be added to the chain
        • E site (exit site) site where discharged tRNA minus amino acids leave ribosome
    • Figure 17.15 Translation – the basic concept
    • Preparation of Eukaryotic mRNA
      • RNA splicing- a cut and paste job to remove nucleotides from transcribed mRNA
        • 8000 nucleotides transcribed but the average gene contains 1200+ nucleotides
        • Long non-coding segments (introns) interspersed between coding segments (exons) expressed via amino acids
    •  
    • Figure 17.17 The initiation of translation
    • Figure 17.18 The elongation cycle of translation
    • Protein Synthesis (cont’) Initiation – elongation - termination
      • Starting at one end of the mRNA, the small ribosomal subunit associates with the mRNA and accepts the first tRNA with its activated amino acid attached = Initiation
      • tRNA associate with a triplet codon exposed on the mRNA – these are 3 nitrogenous bases that bond with 3 complementary bases exposed (anticodon) on the tRNA opposite the attached amino acid
      • Wobble
        • Aren’t 61 tRNAs, are 54tRNAs
    • Figure 17.4 The dictionary of the genetic code
    • Figure 17.3 The triplet code
    • tRNA complexes with its amino acid in the cytoplasm using ATP – activated tRNA
      • The activated tRNA-amino acid complex moves towards the ribosomal area and finds a triplet codon exposed that is complementary to the anticodon of the tRNA
      • The first activated tRNA-amino acid, after its anticodon is bound to the mRNA codon, associates with the large ribosomal subunit which now joins the smaller subunit and the mRNA and the tRNA (TAKE A BREATH!)
    • The first tRNA and its amino acid now occupy the P site of the large ribosomal subunit
      • Review – at this point the 2 part ribosome is assembled, the mRNA has started to be read, and one tRNA plus amino acid is occupying the P site
      • That means the adjacent A site is free to accept a second activated tRNA and its amino acid, but only if the anticodon of this tRNA matches the next three base pairs exposed (codon)
    • Protein Synthesis (continued)
      • At this point, there are 2 tRNA-amino acid complexes adjacent to each other – Elongation involved one amino acid being added in a three step process:
        • Codon recognition – the mRNA codon in the A site matches with the anticodon of the tRNA –amino acid complex
        • Peptide bond formation between the new amino acid in the A site and the amino acid (later peptide) in the P site
        • Translocation
    • Translocation – the ribosome moves the tRNA into the A site, and its attached peptide to the P site, as the previous tRNA from the P site moves to the E (Exit) site and leaves the ribosome
      • Review: once this process is under way, an activated tRNA with its amino acid finds an exposed codon in the A site, attaches via H-bonds, then forms a peptide bond with the polypeptide associated with the tRNA sitting in the adjacent P site. For a moment, the longer polypeptide chain is only attached to the tRNA in the A site. Now the entire ribosome shifts so that the………
    • Yet More Protein Synthesis
      • The empty tRNA from the P site moves in to the E site and leaves the ribosome
      • As the tRNA with the polypeptide chain moves from the A site to the now empty P site ….exposing a new codon.
        • GUESS WHAT HAPPENS NEXT?!
    • A question?
      • Every time a new codon is exposed in the A site, a specific tRNA-AA complex moves into the site. What originally determined this mRNA Codon?
    • The Answer!
      • The original DNA that was transcribed
      • This elongation of 1 AA takes about 0.1 s
      • Termination – the above continues (dozens to hundreds or more AA added) until the STOP CODON is reached (codon at the end of the mRNA)
      • This codon does not have a matching tRNA anticodon so the tRNA-AA attaches in the A site and the tRNA moves to the E site and releases the polypeptide chain
    • FINALLY - SUMMARY
      • The take home message:
        • At the ribosome, the genetic language of DNA is translated into a different language – Via RNA – into the functioning language of PROTEINS!!!!
    • Figure 17.17 The initiation of translation
    • Figure 17.18 The elongation cycle of translation
    • Figure 17.19 The termination of translation
    • Figure 17.20 Polyribosomes
    • Table 17.1 Types of RNA in a Eukaryotic Cell
    • Figure 17.23 The molecular basis of sickle-cell disease: a point mutation
    • Figure 17.24 Categories and consequences of point mutations: Base-pair insertion or deletion
    • Figure 17.24 Categories and consequences of point mutations: Base-pair substitution
    • Figure 17.25 A summary of transcription and translation in a eukaryotic cell
    • Figure 18.19 Regulation of a metabolic pathway
    • Control of Protein Synthesis Regulation of Gene Expression
      • Every cell has the same numbers and types of chromosomes
      • Development and normal gene function requires precise gene expression in an on and off manner
      • Operon – cluster of gene segments on DNA and its controlling segments
        • Repressible
        • Inducible
    • Regions of the Operon (DNA)
      • Promoter region : promotes transcription by binding with RNA polymerase
      • Operator region : binds a regulatory protein or chemical
        • Overlaps with the RNA polymerase binding site
      • Structural genes : code for a particular peptide or several peptides
        • Start or stop codes
    • Figure 18.20a The trp operon: REPRESSIBLE
    • Figure 18.21a The lac operon: INDUCIBLE
    • Figure 19.7 Opportunities for the control of gene expression in eukaryotic cells