Bio3400 Chapter 14 Translation and Proteins
  1. Translation requires        acids,            RNA (mRNA), ribosomes, and           RNA ( tRNA ).

      A prokaryote ribosome consists of a large and a small subunit. Both subunits consist of one or more molecules of ribosomal RNA (rRNA) and several ribosomal proteins. When the two subunits are associated with each other in a single ribosome, the structure is sometimes called a monosome.


      This X-ray diffraction model of a bacterial ribosome shows a small subunit and a large subunit, each with one or two rRNA molecules and 21 - 31 proteins. Three transfer RNAs are bound, interacting with the rRNA of the subunits.


      Eukaryote ribosomes are structurally similar to those of prokaryotes, consisting of a large and a small subunit, each composed of rRNA and ribosomal protein molecules. Eukaryote versions are in general larger than prokaryote ones, and may be found either free-floating in the cytoplasm or associated with the endoplasmic reticulum.


      A tRNA molecule contains many modified bases and contains a series of stems and loops, folded into a 3-dimensional conformation. The 3' end is the amino acid binding site, while the anticodon loop recognizes a codon on the mRNA. The anticodon (CGI) of this tRNA^ala molecule specifies the amino acid alanine, and can base pair with the triplets GCU, GCC, and GCA due to wobble. 2


      May tRNA nucleotides contain nitrogenous bases that are modified aftter transcription. Due to wobble, I (Inosine) and I^m can base pair with U, C, or A.




      In many cases, the first two letters of the genetic code are more critical in specifying an amino acid. For example, the codon for valine (val) only depends on the first 2 letters (GU). The 3rd position of the codon can "wobble": a single tRNA can pair with more than one codon in mRNA. U at the 1st position (5') of the tRNA anticodon may pair with A or G at the 3rd position (3') of the mRNA codon, and G may likewise pair with U or C. Inosine (I), a modified base found in tRNA, may pair with C, U, or A.
     
     
     
     
  2. Each tRNA is "charged" with a specific amino acid by 20 different aminoacyl tRNA              .

      tRNA "charging" starts when an aminoacyl tRNA synthetase catalyzes the conversion of an amino acid to aminoacyladenylic acid. A hydrolyzed ATP donates the phosphate to form the complex. The amino acid is transferred to the appropriate tRNA at the 3' end.
     
     
     
     
  3. Translation of mRNA can be divided into three steps.
       
       
       
       
    •             requires the        and        ribosomal subunits, GTP, initiator tRNA, and initiation          .

        Initiation of translation in E. coli involves the ribosome, mRNA, the energy carrier GTP, several initiation factors ( IFs ), and a tRNA that carries the anticodon UAC and is charged with the modified amino acid N-formylmethionine ( f-met ). Note the E, P, and A sites on the ribosome.


        Translation initiation.
      • The small ribosomal subunit binds to several initiation factors (IF1, 2, 3); this complex binds to mRNA.
      • Initiator tRNA^fmet binds to mRNA AUG codon in the P (peptidyl) site GTP is hydrolyzed to provide energy for the reactions.
      • Large subunit binds to complex. Elongation factor Tu binds to the next tRNA, facilitating entry into the A (aminoacyl) site.
       
       
       
       
    •             requires both ribosomal subunits assembled with the mRNA to form the    site and    site.

        Translation elongation: step 1. Second charged tRNA enters A site, facilitated by EF-Tu. step 2. step 3. step 4. step 5. step 6.


        Translation elongation: step 2. Peptidyl transferase catalyzes the formation of a peptide bond that links the two amino acids. Uncharged tRNA moves to the E site (exit); the mRNA is translocated three bases, resulting in the tRNA bearing the dipeptide to shift into the P site. step 3.


        Translation elongation: step 3. The first link is complete and the first uncharged tRNA is removed from the E site, facilitated by EF-G. The third charged tRNA is ready to enter the A site. step 4.


        Translation elongation: step 4. Third charged tRNA enters the A site, facilitated by EF-Tu. step 5.


        Translation elongation: step 5. The second link is made, forming a tripeptide which starts to emerge through a tunnel in the large subunit. The second uncharged tRNA enters the E site, ready to be removed. step 6.


        Translation elongation: step 8. The whole polypeptide chain is synthesized and released from ribosome when one of the termination codons is encountered.
       
       
       
       
    •              is signaled by a       codon (UAG, UAA, UGA) in the A site.


      Termination of protein synthesis is signaled by a stop codon in the A site: UAG, UAA, or UGA. These codons do not bind a tRNA in the A site, so the A site is empty. GTP-dependent release factors cleave the polypeptide chain from the terminal tRNA in the P site, releasing it from the translation complex, which dissociates.
     
     
     
     
  4. A mRNA molecule can have several            simultaneously translating the message, forming                ( polysomes ).

      Polysomes from rabbit reticulocytes (immature red blood cells) engaged in the translation of hemoglobin mRNA. Polysomes from salivary gland cells of the midgefly, Chironomus thummi; polypeptide chains can be seen emerging from the ribosomes.
     
     
     
     
    Review: interactive polypeptide coding, translation steps, animation.
     
     
     
     
  5. Studies of mutations that result in heritable human diseases such as               and           provided first insight into the role of proteins in genetic processes.

      Metabolism of the amino acids phenylalanine and tyrosine require several enzymes. Various metabolic blocks due to mutations lead to defective enzymes and the disorders phenylketonuria, tyrosinemia, albinism, and alkaptonuria.
     
     
     
     
  6. Studies of mutations in Neurospora led to the one-gene:one-         hypothesis.

      Beadle and Tatum (Nobel 1958) induced nutritional auxotrophic mutations in the bread mold Neurospora by exposing asexual conidia (spores) to radiation. next, conclusion.


      By attempting to grow the mutants on minimal media supplemented with various organic compounds, the nutritional mutation can be pinpointed. next


      Beadle and Tatum were able to determine that each genetic mutation is associated with a loss of the enzymatic activity to synthesize a vitamin or an amino acid. This led them to propose the hypothesis that one gene specifies one enzyme. (Now we know not all proteins are enzymes and some proteins have more than one polypeptide chain.)
     
     
     
     
  7. Studies of human             showed that one gene encodes one              .


        Normal erythrocytes (red blood cells) have a biconcave disc shape. They contain hemoglobin molecules that appear red when oxygenated. The recessive genetic disorder sickle-cell anemia results in abnormal hemoglobin molecules that cause erythrocytes to become elongated and curved under low oxygen levels.


        Fingerprint analysis The protein is digested into peptide fragments by proteolytic enzymes. The mixture is then subjected to paper electrophoresis followed by paper chromatography at 90 to the electrophoresis. The resulting two-dimensional "fingerprint" reveals that normal hemoglobin HbA and sickle-cell HbS hemoglobin differ by a single peptide fragment. Further analysis shows a single amino acid change: valine is substituted for glutamic acid at the 6th position of the beta chain.
       
       
       
       
    • Humans possess        different hemoglobin polypeptide genes; two of which are expressed at different times in development


      A variety of hemoglobin molecules are produced in humans at different stages of life. All are tetramers consisting of seven distinct polypeptide chains, each encoded by a separate gene. Embryonic hemoglobin has z (zeta) and e (epsilon) chains. By eight weeks gestation, these are replaced by fetal hemoglobin with alpha chains and two types of g (gamma) chains. 98% of adult hemoglobin are HbA molecules consisting of alpha and beta chains; 2% are HbA2, composed of alpha and d (delta) chains.
     
     
     
     
  8. The order of nucleotides in a gene is           with the order of amino acids in the corresponding polypeptide.

      Colinearity in the trpA gene that encodes the A subunit of the enzyme tryptophan synthetase in E. coli is shown by comparing the gene maps of several mutants and the altered amino acid positions in each mutant protein.
     
     
     
     
  9. Amino acids are composed of a           group, an        group, and an R group bound to a central         atom.

      Amino acids are composed of a carboxyl group, an aminoamino group, and an R group bound to a central carbon The R (radical) group confers specific chemical properties to each amino acid. The R groups can be divided into four main classes:
    • nonpolar (hydrophobic)
    • polar (hydrophilic)
    • negatively charged
    • positively charged


      These amino acids have R groups composed only of carbon and hydrogen atoms, making them nonpolar and hydrophobic. Pro has a nitrogen atom and is sometimes classified as polar. Met has a sulfur atom but remains hydrophobic while phe and trp are sometimes classified as aromatic, since they have a benzene-like ring.


      These amino acids have oxygen, sulfur and/or nitrogen in the R group, making them polar and hydrophilic. They are electrically neutral molecules. Gly is sometimes classified as nonpolar and tyr is sometimes classified as aromatic.


      These amino acids are carry a negative charge, making them acidic. These very polar molecules are highly hydrophilic.


      These amino acids are carry a positive charge, making them basic. These very polar molecules are highly hydrophilic.
     
     
     
     
  10. The          structure of a polypeptide is the sequence of amino acids formed by linking them together by          bonds.

      A covalent peptide bond is formed when the amino group of one amino acid reacts with the carboxyl group of another amino acid by a dehydration (condensation) reaction, releasing a molecule of H[2]O. The sequence of amino acids constituting a polypeptide is called its primary structure.
     
     
     
     
  11. Proteins may also exhibit        additional levels of structure: secondary, tertiary, and quaternary.

      Hydrogen bonding in a regular, repeating pattern stabilizes sections of a polypeptide, forming an alpha-helix secondary structure. Keratin , the tough structural protein of hair, is rich in alpha helix. continue


      Hydrogen bonding may also form a beta-pleated-sheet secondary structure in a zigzagging plane. Extensive beta-pleated-sheet regions provides strength and rigidity to structural proteins such as the fibroin found in silk.


      The secondary structure sections of a polypeptide may further be organized into a tertiary structure by these interactions:
    • Covalent disulfide bonds form between polar cysteine molecules.
    • Most of the polar hydrophilic R groups are located on the surface, close to surrounding water molecules.
    • Most nonpolar hydrophobic R groups are located on the interior, avoiding water. This diagram shows tertiary structure of myoglobin, an oxygen carrier molecule in muscle cells.


      Some proteins, especially enzymes, are oligomeric: composed of more than one polypeptide chain. Hemoglobin is an oligomeric protein consisting of four protomers (subunits): two alpha and two beta chains that fit together with four heme groups in a quaternary structure comprising the functional molecule.
     
     
     
     
  12. Proteins play diverse roles in the body.
       
       
       
       
    • Hemoglobin binds to and transports         , which is essential for cellular metabolism.

        Normal erythrocytes (red blood cells) have a biconcave disc shape. They contain hemoglobin molecules that appear red when oxygenated. The recessive genetic disorder sickle-cell anemia results in abnormal hemoglobin molecules that cause erythrocytes to become elongated and curved under low oxygen levels.


        Fingerprint analysis The protein is digested into peptide fragments by proteolytic enzymes. The mixture is then subjected to paper electrophoresis followed by paper chromatography at 90 to the electrophoresis. The resulting two-dimensional "fingerprint" reveals that normal hemoglobin HbA and sickle-cell HbS hemoglobin differ by a single peptide fragment. Further analysis shows a single amino acid change: valine is substituted for glutamic acid at the 6th position of the beta chain.
       
       
       
       
    • Collagen and keratin are             proteins.

        Collagen is a fibrous structural protein that has left-handed helical subunits intertwined into a larger triple helix.

        This structure provides flexible strength to connective tissue in skin, tendons, and ligaments in animals.

       
       
       
       
    • Actin and myosin are              proteins in muscle tissue.
       
       
       
       
    •                  ( antibodies ) recognize "foreign" particles in the immune system.
       
       
       
       
    • Transport proteins move molecules across            .

        Facilitated diffusion: carrier proteins. A carrier protein alternates between two conformations, moving a solute across the membrane as the shape of the protein changes. The protein can transport the solute in either direction, with the net movement being down the concentration gradient of the solute.
       
       
       
       
    • Some           and their receptors are proteins.
       
       
       
       
    •           bind to DNA in eukaryotic organisms.
       
       
       
       
    • Enzymes act as            in biological reactions.


      Enzymes act as catalysts by lowering the energy of activation in cellular reactions, thus speeding up the chemical reactions. As catalysts, the enzymes are not part of the chemical reactions themselves, and can be re-used after the reaction.