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Lecture 27: Gene to Protein

  1. Information Flow: The Central Dogma
    1. Francis Crick provided the notion and much of the experimental evidence
    2. Biological information flows from DNA to RNA to protein and does not flow back
    3. Specific RNA molecules, called transfer RNAs (tRNA), provide the transition between the sequence of bases in nucleic acid and the sequence of amino acids in proteins.
    4. Another class of RNA molecules, messenger RNAs (mRNA), are made as complements to the DNA of genes coding for proteins and are transported to the cytoplasm for protein synthesis

  2. Transcription
    producing RNA single strands from the DNA

    1. requires a double stranded DNA template and the ribonucleoside triphosphates (ATP, GTP, CTP, and UTP)
    2. carried out by an RNA polymerase which creates a single-strand RNA complementary to one of the DNA strands (sense or template strand)
    3. produces four categories of RNA
      1. rRNA or ribosomal RNA (1,700 to 4,000 bases) (80% of E. coli RNA)
      2. tRNA or transfer RNA (75 to 120 bases) (15% of E. coli RNA)
      3. mRNA or messenger RNA (variable size) (5% of E. coli RNA)
      4. other
    4. RNA polymerase is oligomeric and has a subunit which recognizes a binding site on the DNA
    5. RNA strand is released from binding with the template DNA during synthesis
    6. four processes
      1. RNA pol binding - bind at DNA sequences called promoters
        • some promoters are more efficient than others which leads to differential gene expression
        • DNA helix is opened up
      2. initiation
        • binding of first base - a purine nucleotide triphosphate
        • this step is blocked by the drug Rifamycin
      3. elongation - at about 20 to 50 nucleotides per second
        • bind the next nucleotide triphospate as determined by the DNA sequence in the 3'-5' direction of the template strand
        • form a covalent bond between the alpha phosphate of the second base and the 3' OH of the first base
        • repeat
      4. termination - protein assisted or sequence assisted
        • some proteins exist that recognize certain sequences in RNA and cause a release of the RNA chain from the DNA and RNA pol
        • a sequence in the 3' end of the RNA may cause it to pair on itself which results in termination
    7. prokaryotes have one RNA polymerase with several, different promoter binding subunits
    8. eukaryotes have three, different RNA polymerases that read specific classes of genes
      1. RNA Pol I - localized to the nucleolus and reads ribosomal RNA genes (rRNA)
      2. RNA Pol II reads genes that code for proteins or messenger RNA (mRNA)
      3. RNA Pol III transcribes transfer RNA genes (tRNA), the genes for a small ribosomal RNA and some other small RNA molecules
      4. eukaryotes also have a variety of transcription factors that are involved in promoter binding and transcriptional regulation

    9. The Genetic Code

      1. 3 consecutive bases code for one amino acid
      2. code is nonoverlapping and commaless
      3. code is degenerate - some amino acids have more than one code (arg, leu, ser each have 6 codes)
      4. 1st and 2nd bases more important than the 3rd base;"wobble" in the 3rd position allows the cell to"get by" with fewer than 61 different tRNA species
      5. 3 codes are used for STOP punctuation (UGA, UAG, and UAA)
      6. 1 code is used for translation initiation (AUG = methionine)
      7. code is nearly universal
      8. not all codes for an amino acid are used at the same frequency
      9. fidelity resides in enzymatic attachment of specific amino acid to the tRNA with the appropriate anti-codon
        • there is an aminoacyl-tRNA synthetase for each amino acid
        • the amino acid is attached to the 3'OH of the last base of the tRNA
        • the reaction occurs in two steps
        • the first step utilizes ATP and results in the formation of an amino acid adenylate and the release of pyrophosphate
        • the second step is the transfer of the amino acid to the tRNA
        • the fact that two recognition steps are required leads to a higher fidelity of code translation

    10. Ribosomes

      1. consist of two subunits of differing size and composition - a small subunit and a large subunit
        CategorySubunit SizerRNA
        Present        		Proteins
        Present
        Prokaryoticsmall30S16S21
        large50S23S, 5S31
        Eukaryoticsmall40S18S33
        large60S28S, 5.8S, 5S49
      2. one function for the rRNA is as a complementary match against certain sequences in the other RNAs during protein synthesis
      3. the largest ribosomal RNA of the larger subunit may carry out the peptidyltransferase function - a nucleic acid functioning as an enzyme
      4. the ribosomal proteins function in a variety of ways for ribosomal structure, recognition of mRNA and tRNA, etc.

    11. Translation: Synthesis of Polypeptide Chains

      1. initiation
        1. form an initiation complex consisting of
        2. small ribosomal subunit which recognizes a sequence of bases in the 5' end of the message near the start AUG
        3. the 5' end of a mRNA at the site of the initiator codon (AUG)
        4. an activated, initiator tRNA with attached amino acid
          • prokaryotes the initiator tRNA carries a methionine which has a modified amine group such that it can form a peptide bond in only one direction
          • eukaryotes use an unmodified methionine for initiation but use a specific tRNA for initiation
        5. requires protein initiator factors that are activated by the the attachment of GTP
        6. match the anticodon of the initiator tRNA to the AUG start codon of the mRNA
        7. bind the large ribosomal subunit such that the bound initiator tRNA is located in the P site (peptidyl) of the subunit
          • large ribosomal subunit has two general sites
          • the P site holds the tRNA with the attached polypeptide chain
          • the A site holds the tRNA with the next amino acid to be attached to the growing polypeptide chain
        8. release the initiator factors as the bound GTP is cleaved and removed to become GDP and Pi

      2. elongation

        1. bind in the A site an activated tRNA containing an anticodon complementary to the next three bases from the initiator codon proceeding in the 5' to 3' direction along the mRNA
        2. this binding is assisted by a protein elongation factor (EF-Tu) that has been activated by the attachment of GTP
        3. the elongation factor is released with a cleavage of GTP
        4. the peptide bond is formed between the carboxy group of the amino acid in the P site and the amino group of the amino acid in the A site
        5. peptide bond formation is carried out in the presence of an enzyme peptidyltransferase
        6. at this point, the polypeptide chain is attached to the tRNA in the A site
        7. a second, GTP activated elongation factor (EF-G) is bound
        8. the de-activated tRNA in the P site is released
        9. the mRNA and tRNA with attached polypeptide chain in the A site are translocated to the P site
        10. the EF-G factor is released
        11. the process repeats

      3. termination and release

        1. every time a new codon is brought into the A site, it is tested for termination by the presence of release factors
        2. if one of the termination codons (UGA, UAG, or UAA) is brought into the A site, a release factor is activated
        3. the activated release factor causes peptidyl transferase to transfer the polypeptide chain to a water molecule
        4. this releases the polypeptide chain from the tRNA and from the ribosome
        5. the unactivated tRNA is released from the P site
        6. the ribosome releases into its two subunits ready to be utilized again
      4. Summary (animation .mov)

    12. Regulation of Translation

      1. polysomes
        • once a ribosome has moved away from the initiation area, another initiation complex can form
        • mRNA molecules with multiple ribosomes are termed polysomes
        • polysomes increase the effectiveness of an mRNA
      2. prokaryotes (no nuclear membrane to separate the DNA from the cytosol) carry out translation on mRNA as it is being produced from the DNA - coupled transcription and translation
      3. prokaryotes often have a mRNA containing the information for more than one polypeptide
        the genes are contiguous on the DNA - polycistronic message (cistron = gene)
      4. prokaryotic mRNAs have a half-life on the order of minutes
      5. eukaryotic mRNAs often have a half-life of days
      6. antibiotics - chemical warfare among organisms
        Prokaryotes
        AntibioticEffect on Translation
        StreptomycinPrevents formation of initiation complex
        TetracyclineInhibits tRNA binding at A site
        ErythromycinBlocks peptidyl transferase activity
      7. signal sequence marks a polypeptide chain for export

    13. Mutation Types and Effects

      1. Point mutations at the DNA level
        • base replacement leads to silent mutant or missense
        • some base replacement will lead to nonsense and early termination
        • base addition or deletion leads to a shift in reading frame (frameshift) which results in missense followed by a nonsense
        • examples in the globin gene

      This document maintained by Robert J. Huskey.
      Last update on November 2, 1999.