Topic 13, Translation and Proteins

In translation, the language of mRNA is translated into the language of proteins

  1. Ribosome structure
    A. Ribosomes are small bodies that contain about 90% of the RNA in the cell
         (one bacterial cell contains ~10,000 ribosomes)
    B. Ribosomes are the sites of protein synthesis in the cell
    C. Ribosomes have two subunits, a large subunit and a small subunit   
        1. In prokaryotes, there are 50S and 30S subunits
            a. 50S subunit has 23S rRNA, 5S rRNAs, and 31 proteins
            b. 30S subunit has 16S rRNA and 21 proteins
            c. The ribosomal subunits are capable of self-assembly
        2. In eukaryotes, there are 60S and 40S subunits
            a. 60S subunit has 28S, 5.8S, and 5S rRNAs and 49 proteins
            b. 40S subunit has 18S rRNA and 33 proteins
        3. The ribosomes are ~1/2 protein and ~1/2 RNA
        4. A single precursor molecule encodes all 3 rRNA sequences in E. coli and there are
            7 copies of this sequence in the genome
        5. A single precursor molecule encodes the 18S, 28S, and 5.8S rRNAs in eukaryotes,
            the 5S is encoded elsewhere in the genome
            a. All of the precursor molecules that encode the 18S, 28S, and 5.8S rRNAs are at the
                nucleolar organizing region
            b. There are hundreds to thousands of these precursor molecule templates per haploid genome
            c. The nucleolar organizing region is necessary to organize the nucleolus,
                no nucleolus is present if the NOR is absent
            d. The ribosomal subunits are made in the nucleolus in eukaryotic cells

  2. tRNA structure
    A. tRNAs are 75-90 nucleotides long, 4S
    B. Transcribed as larger precursors and cleaved into mature 4S molecules
    C. Holley et al. (1965) determined the complete nucleotide sequence of the first tRNA
    D. The two-dimensional model has
         1. An amino acid binding site (CCA sequence which binds the amino acid)
         2. Anticodon loop
         3. D-loop and T-pseudouracil loop and a variable loop
         4. It has several regions of paired bases and has the shape of a cloverleaf
         5. It contains many unusual bases
    E. A three-dimensional model, given in Fig. 14.4

  3. Charging the tRNA (attaching the amino acid to the tRNA
    A. Aminoacyl-tRNA synthetases or simply synthetases attach the correct amino acid to the
         correct tRNA in a two-step process (see handout for details)
        1. Amino acid activation
        2. Charging the tRNA with an amino acid --> aminoacyl-tRNA

  4. Translation
    A. Initiation
        1. Initiation complex - 3 components join together to form the initiation complex
            a. Small ribosomal subunit
            b. Chain initiation codon in mRNA (AUG)
            c. First tRNA attaches to the P (pepidyl-tRNA binding) site on the small ribosomal subunit
            d. The first AUG codon codes for formly methionine in prokaryotes and methionine in eukaryotes.
                Subsequent AUG codons code for methionine in both prokaryotes and eukaryotes.
    B. Elongation
        1. The large ribosomal subunit joins the initiation complex
        2. There are 3 major steps in elongation
            a. A second aminoacyl-tRNA binds to the next codon at the A site
            b. A peptide bond forms between the amino acids
            c. Translocation
                1) The ribosome moves one codon in relation to the mRNA
                2) The tRNA moves from the A site to the P site on the ribosome
                3) The tRNA is released from the P site on the ribosome
        3. A tunnel exists within the large subunit through which the elongating polypeptide emerges
    C. Termination
        1. The chain termination codons are UAA, UAG, and UGA
        2. When a chain termination codon is reached, the large and small subunits of the ribosome dissociate
        3. The polypeptide chain and the mRNA are released from the ribosome
    D. Therefore, the sequence of amino acids in a protein is determined by the sequence of nucleotides
         in the DNA

  5. Polyribosomes (=polysomes)
    A. Several ribosomes are attached to each mRNA which simultaneously transcribe the mRNA molecule

  6. Differences in translation in prokaryotes and eukaryotes
    A. Eukaryotic mRNA is much longer-lived (hours) than prokaryotic mRNA (minutes)
    B. The 5' end in eukaryotes is "capped" with a 7-methylguanosine residue and
         a poly-U tail is added to the 3' end
    C. The first amino acid is methionine in eukaryotes and formylmethionine in prokaryotes

  7. Garrod's initial evidence that genes are involved in protein production
    A.Garrod (1902, 1908), an English physician, was interested in human diseases that might have
         a genetic basis
    B.He studied the inheritance of alkaptonuria and concluded that it was due to an
        autosomal recessive mutation
    C. Characteristics of alkaptonuria
        1. The affected individual's urine turns black when exposed to air
        2. The cartridge in the ears and nose turns black and the whites of the eyes turn black
        3. These are all due to the accumulation of alkapton (homogentisic acid)
    D. Garrod fed affected individuals chemicals which are related to alkapton (phenylalanine or tyrosine)
        and found that the amount of alkapton in the urine increased.  However, when he fed these same
        compounds to normal individuals, there was no increase of alkapton in the urine.
    E. Garrod concluded that there was a genetic block in the metabolism of homogentisic acid.
    F. Garrod hypothesized that hereditary information controls chemical reactions in the body
    G. Perhaps best stated, one gene-one metabolic block

  8. Beadle and Tatum's work provided the first convincing evidence that genes are directly responsible for the production of proteins
    A. Beadle and Tatum produced the first biochemical mutants (in Neurospora crassa, the bread mold)
        1. Neurospora will grow on a defined minimal medium, Fries medium + biotin
            a. Minimal medium only contains the nutrients that are necessary for growth of a wild-type organism
                1) If you remove any one component of the minimal medium, the organism cannot grow or
                    grows poorly
            b. Complete medium is minimal medium supplemented with other nutrients
        2. Beadle and Tatum X-rayed conidia (asexual spores) to increase the mutation frequency and
            plated them on complete medium.
            a. They then transferred cells of each colony which grew onto minimal medium and
                determined which ones would not grow.
                1) The ones which would not grow were auxotrophs (nutritional-requiring mutants)
                2) Ones that could grow on minimal medium are referred to as prototrophs
            b. They then placed the prototrophs individually on minimal medium which was supplemented with
                various classes of compounds (e.g., amino acids, vitamins, purines and pyrimidines, etc.) to
                determine which class of compounds was necessary for growth of each mutant.
            c. Once they determined the class of compound which was affected in each mutant, they added the
                individual members of that class of compound one at a time to determine the specific one that
                was necessary for growth.
            d. For example, if a auxotroph was not able to grow in minimal medium but able to grow in
                medium that was supplemented with amino acids, they concluded that the organism was
                not able to produce at least one of the amino acids.
            e. They then added the amino acids to minimal medium one at a time to determine which amino
                acid the organism could not produce
        2. They isolated hundreds of biochemical mutations and found that each mutation had
            a. A single nutritional requirement and
            b. A mutation in only one gene
        3. These observations led Beadle and Tatum to conclude that one gene specifies one enzyme,
            the one gene : one enzyme hypothesis
        4. Beadle and Tatum were awarded the Nobel prize for this work in 1958

  9. Use of auxotrophs for working out biochemical pathways (Srb and Horowitz, 1944)
    A. They isolated 7 auxotrophs in Neurospora crassa which each required arginine for growth
    B. They added arginine or presumptive precursors of arginine (ornithine or citrulline) to minimal medium
        and placed each auxotroph on the medium
        1. One mutant grew if arginine was added but not if ornithine or citrulline was added (arg-1 mutation)
        2. Two mutants grew if arginine or citrulline were added but not if ornithine was added
            (arg-2 and 3 mutations)
        3. Four mutants grew if arginine, citrulline, or ornithine were added (arg 4, 5, 6, and 7 mutations)
    C. From these observations, they concluded that a precursor was converted to ornithine which was
         converted to citrulline which was converted to arginine (see notes and/or book for details)
    D. The mutants within each group mapped at a single locus and each group of mutants mapped to
        different  loci
    E. In lab, we are working with different yeast mutations requiring adenine
        1. Two adenine-requiring mutations were crossed together and plated on minimal medium.
            a. If the diploid progeny cells grew on minimal medium, we would conclude that the two mutations
                are not allelic (do complement)
            b. If the diploid progeny cells do not grow on minimal medium, we would conclude that the two
                mutations are allelic (do not complement)

  10. A few examples of biochemical mutants in humans
    A. Phenylketonuria = PKU
        1. Due to a deficiency in the enzyme that converts phenylalanine to tyrosine (occurs in the liver)
        2. Autosomal recessive, occurs in 1/1100 live births
        3. Causes severe mental retardation if not treated
        4. The symptoms are due to the accumulation of phenylalanine
        5. The developing babies with PKU are normal prior to birth because excess phenylalanine is
            removed by the placenta
        6. Within 3 days after birth, the level of phenylalanine goes up sharply in babies with PKU
        7. If individuals with PKU are placed on a diet that is low in phenylalanine for the first 6-9 years,
            their development is relatively normal.  After this, they can have a normal diet because the brain is
            fully developed by this time.
        8. All newborns in the US are tested for PKU
    B. Cretinism (due to an autosomal recessive)
        1. An enzyme which is needed for synthesis of thyroxin by the thyroid gland is defective in individuals
            with this genetic disease
        2. Fetal development is normal because the hormone comes from the mother
        3. A few weeks after birth, there is mental dullness and retarded growth
        4. If thyroxin (thyroid hormone) is administered, development is normal
    C. Albinism
        1. Due to an autosomal recessive
        2. An enzyme is not produced which is necessary for the synthesis of melanin
    D. Tyrosinosis
        1. Due to an autosomal recessive
        2. Harmless condition in which the individual has an excess level of tyrosine in the blood
    E. Alkaptonuria, already discussed
    F. Therefore, there are a large number of molecular diseases in this small biosynthetic pathway
    G. Galactosemia
        1. Due to an autosomal recessive
        2. 1/57,000 births
        3. Enlarged liver, vomiting, cataracts, mental retardation, failure to thrive (sickly)
        4. Due to the lack of a liver enzyme, GPT (galactose 1-phosphate uridyl transferase) which converts
            galactose-1P to glucose-1P
        5. The accumulation of galactose-1P causes the problem
        6. If the infant is fed milk, the major carbohydrate in milk is the dissacharide, lactose, which is a
            glucose-galactose disaccharide
        7. If detected at birth, the individual can be fed a diet that lacks lactose to overcome this genetic
            defect.
    H. Enzymatic pathways
        1. A precursor, A is converted by the action of an enzyme (enzyme A) to endproduct B
        2. If the enzyme is defective or missing, there will be an increased level of compound A and a
            decreased level of endproduct B.
        3. For some genetic conditions, the accumulation of the precursor causes the problem
            a. Examples: galactosemia, PKU, alkaptonuria
        4. For other genetic conditions, the lack of an endproduct causes the problem
            a. Examples: cretinism and albinism
        5. Thousands of human genetic diseases are now known, and most are due to an alteration in a single
            gene.  Many of these can be treated.

  11. Studies of human hemoglobin established that one gene encodes one polypeptide
    A. Sickle cell anemia characteristics
        1. Discovered in 1910 and inherited as a Mendelian recessive
        2. Red blood cells (erythrocytes) in affected individuals assume a distinctive sickle shape under
            low oxygen conditions
        3. These sickle cells plug capillaries resulting in oxygen loss to tissues and the individual
            experiences sickle-cell crisis.  Can be fatal.
        4. Controlled by a single locus, HbA is the normal allele and HbS is the mutant allele
    B. Linus Pauling (1949) found that hemoglobin molecules from normal and affected individuals had
        different migration rates when subjected to electrophoresis.  He also found that the heterozygote
        had both forms; thus, this gene exhibits codominance
    C. Hemoglobin molecules have 4 iron-containing heme groups (non-protein) and 4 polypeptide chains
        (2 alpha globin chains and 2 beta globin chains)
    D. Ingram (1954-1957)
        1. Cut the alpha globin chains and beta globin chains into small pieces (peptide fragments) and
        2. separated the peptide fragments from each other by 2-dimensional electrophoresis
        3. He found that one peptide fragment from the beta globin chain was different in normal and
            sickle-cell individuals
        4. Also found that there was a single amino acid substitution in this peptide fragment
            a. Normal had glutamic acid and sickle cell had valine
        5. This was the first demonstration that a mutation that affects a phenotype can be due to a
            single amino acid substitution

  12. Colinearity of a gene and a polypeptide (Yanofsky 1967)
    A. He recovered many different mutations in the tryptophan synthetase gene in E. coli
    B. He determined where each of the mutations caused an amino acid change in the
        tryptophan synthetase protein
    C. He also mapped each of the mutations within this gene
    D. He found that the order of alterations in the polypeptide was the same as the
        order of mutations in the genetic map: therefore,
        the a gene and a polypeptide are collinear

  13. Protein chemistry. 
    I expect that each of you have had this in other courses and I expect you to know important basic information about proteins such as:
    A. There are 20 types of amino acids in living things
    B. The amino acids are attached to each other by peptide bonds (which are covalent bonds)
    C. A chain of amino acids is a polypeptide
    D. There is a N-terminus and a C-terminus and you know what they are
    E. There are 4 levels of protein structure that are recognized,
        1. Primary structure = the sequence of amino acids in a polypeptide
        2. Secondary structure = alpha helix or beta-pleated sheet
        3. Tertiary structure = folding of a protein
        4. Quaternary structure = association of polypeptides with each other
    F. Proteins can be structural or enzymes
        1. Enzymes = a protein or complex of proteins that catalyzes a specific chemical reaction

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Updated 11/28/00