Biochemistry I Fall Term, 2000

 October 25, 2000

Lecture 22: Protein Synthesis: Ribosomes & Peptide Bond Formation

Assigned reading in Campbell: Chapter 8.8-8.11

Key Terms:
30S and 50S ribosomal subunits
16S and 23S rRNA
Comparative sequence anaylsis
Reconstitution of functioning ribosomes
A site -> P Site -> E Site 		Ribosome binding site
Peptidyl transferase center
Translocation
Chloramphenicol and puromycin
 

Take the Review Quiz on Lecture 22 concepts.

Structure of the 50S Ribosome Subunit is a page of images to accompany this lecture.
Polypeptide Elongation Cycles is an animation of polypeptide chain elongation.
Cryo-electron microscope images
The ribosome of Escherichia coli at 25 Å resolution from researchers at the University at Albany
Blueprints of the Protein Factory from the NCSA at University of Illinois

Handout for this lecture: Recap of Gene Expression Lectures (#17-22)

These notes continue the outline from Lecture 21.

3. Ribosome

The ribosome is a protein-synthesizing machine.
A. Ribosome Structure (bacterial)

  1. RNA and protein components.
    a) the ribosomal subunits and their RNA components are named for their sedimentation coefficients (Campbell, pp. 276-268).
      30S subunit 50S subunit
    rRNA 16S 23S and 5S
    Proteins 21 31
    Overall, the 70S ribosome is about 66% RNA by mass.
    Eukaryotic ribosomes: Similar but the RNA's are larger and the proteins are more numerous.
    b) Ribosomal RNA's from all species are homologous, i.e. a single progenitor and diverse lines of descent during their evolution. Certain nucleotides and base pairs are phylogenetically conserved. Some of these are noncannonical "pairs" e.g. G-U and G-A. Secondary (and some tertiary) structures of rRNAs have been determined with comparative sequence analysis (Campbell, Fig. 7.21). If we assume that the structure and function of the ribosome has remained relatively constant through evolution, then the sequences of rRNA's from different species provide a "natural experiment" in nucleotide substitution. For example, a helix is considered to be established if changes in its base pairs are highly correlated, e.g. A -> C at a position covaries with U -> G at that position.
    c) A few 3D structures of individual proteins have been determined.
  2. Reconstitution of functioning ribosomes from purified components.
    a) Nomura and coworkers established the assembly pathway.
    b) It is a mostly ordered pathway in which early binding reactions create the binding sites for subsequent assembly steps.
    The impact of Nomura's work for the study of ribosome structure and function has been enormous. For example, much less is known about eukaryotic protein synthesis simply because it is not yet possible to take the "machine" apart and put it back together again.
  3. Low resolution (20Å) structures of ribsomes have been determined using (computer-assisted) image reconstruction of many cryo-electron microscope images (Links at the top of this page).

B. Polypeptide Synthesis: An Overview.

  1. Translation occurs on polyribosomes (polysomes)
  2. The polypeptide grows stepwise as a ribosome-bound, peptidyl-tRNA.
    Discrete sites on both ribosomal subunits accomodate steps in elongation.
    a) The P site binds the peptidyl-tRNA
    b) The A site binds the aminoacyl-tRNA corresponding to the next amino acid.
    c) The E site is the exit site for the tRNA that was previously in the P site.
    The path of a tRNA through the ribosome is: A site --> P site --> E site.

C. Chain Initiation

  1. N-formylmethionine (fMet) and its charged tRNA function uniquely to initiate chains.
  2. The ribosome binding site on mRNA is defined by the Shine-Dalgarno sequence (SD), a spacer length, and the downstream initiation codon (AUG).
    a) The SD sequence (5'GGAGGU3') was one of the the first "consensus sequences" proposed (1974).
    b) The spacing between SD and AUG affects initiation efficiency.
    c) The SD sequence base pairs to a region at the 3' end of 16S rRNA.
    The agreement between any given ribosome binding site on a mRNA and the [consensus sequence + spacing] correlates well with in vivo protein synthesis efficiencies. Thus, as we saw for RNA synthesis, initiation of the polymerization is the most important control step. Moreover, the template sequences in both cases determine initiation frequency.
  3. Chain initiation requires the transient association of initiation factors and GTP hydrolysis.
    a) mRNA first associates with the 30S subunit.
    b) fMet-tRNAfMet and the 50S subunit combine to form the 70S initiated complex.


D. Chain Elongation

The three states of the ribosome during one round of polypeptide elongation.
Rectangles are the E, P, and A sites on each ribosomal subunit. The vertical lines are tRNAs, colored to match their cognate amino acid. The growing protein chain is shown as a wavy line. (cf. Fig. 9.8 in Campbell.)
At the left, a peptidyl-tRNA occupies the P site. In the first step, EF-Tu with bound GTP, brings the next aminoacyl-tRNA to the A site. Following peptidyl transfer and translocation, the newly elongated peptidyl-tRNA occupies the P site. The first tRNA (green) is now in the E site and will dissociate shortly, returning the ribosome to the start of the cycle.
Not depicted here are the mRNA-anticodon interactions that occur on the 30S subunit. The student is encouraged to draw these on the diagram, e.g. use "aa1", "codon1", etc. instead of the color-coding used here.
(Look at the animation of Polypeptide Elongation Cycles to get a perspective on the sequence of these events.)
  1. The EF-Tu-GTP-aminoacyl-tRNA complex is the active species for charged tRNAs binding to the 70S ribosome.
  2. Transpeptidation (peptide bond formation) joins the growing peptide (in the P site) to the aminoacyl-tRNA in the A site. The protein is now one amino acid longer, but it is bound as peptidyl-tRNA in the A-site.
    Note the following features of peptide bond formation per se:
    a) neither ATP nor GTP is hydrolyzed.
    b) the peptidyl transferase center is on the 50S subunit, and may reside on 23S RNA, itself. (See below.)
    c) transpeptidation is energetically favorable.
  3. Translocation is required to complete an elongation cycle. The peptidyl-tRNA is translocated from the A site to the P site.
    a) concurrently, but perhaps not simultaneously, the now uncharged tRNA moves to the E site and dissociates from the ribosome; and then,
    b) the mRNA, now associated with the anticodon of the next aminoacyl-tRNA, also translocates on the 30S subunit to its P site.
    c) translocation requires EF-G and GTP hydrolysis
    d) intermediate (or hybrid) states have been characterized in which the peptidyl-tRNA has moved to the P site on the 50S subunit, but still occupies the A site on the 30S subunit: i.e. an A/P hybrid.
    Details of the molecular events during the translocation events are obscure. We know the machine moves; many of the moving parts have been identified; we even have some snapshots of stopped motion; but we still do not know whether the motion is continuous, ratchet-like, or something else. For example, one class of models proposes that the ribosome is rigid, and all of the other components move relative to it. An alternative view states that the 30S and 50S subunits rotate relative to one another during each translocation step; in this model, the other components can be viewed as being fixed. Neither extreme model can be excluded.
  4. Peptide bond formation was localized to the 50S ribosomal subunit based on the results of the puromycin reaction. Binding of puromycin in the A-site, followed by peptide bond formation, results in termination of translation because the polypeptide-puromycin cannot be elongated. It was later found that isolated 50S subunits could catalyze the reaction using fMet-tRNAfMet and puromycin in the presence of ethanol or methanol.
  5. Since the summer of 2000, it is clear that 23S rRNA provides all of the catalytic groups for the peptidyl transferase reaction, i.e. the ribosome is a ribozyme. This strong conclusion followed from the 50S subunit structure determination (Structure of the 50S Ribosome Subunit) and biochemical results that localized the active site to a few nucleotides on 23S rRNA. Previous evidence and arguments pointing to this view were:
    a) RNA can act as a catalyst in vivo and in vitro.
    b) Removal of "nearly all" protein does not abolish the peptidyl transferase activity of the remaining rRNA.
    c) Resistance to peptidyl transferase drugs is found in the genes for 23S rRNA.

E. Chain Termination

Release factors, a stop codon (or two), and GTP hydrolysis are required to finish the polypeptide chain.

F. Translational Accuracy

  1. Mistranslation has been measured: about 10-4 errors/codon.
  2. "Kinetic proofreading" is an alternative fidelity mechanism to hydrolytic editing at a second active site, e.g. the aaRS enzymes.

G. Antibiotics
Many antibiotics bind to the ribosome and interfere with protein synthesis at discrete steps in the initiation or elongation phases. The identity of antibiotic resistance genes provided early evidence for a central role of rRNA in protein synthesis. In general, bacteria acquire resistance to a drug by: 1) pumping it out of the cell (e.g. tetracycline); 2) modifying it enzymatically (e.g. chloramphenicol); or 3) mutating the gene for the target molecule. In the latter group, it turns out that most of the mutations to drug resistance occur in the genes for 16S and 23S rRNA, and not in the ribosomal protein genes.

  1. Chloramphenicol inhibits peptidyl transferase.
  2. Puromycin is an analog of the 3' terminus of Tyr-tRNATyr (Campbell, Fig. 9.9).


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