Prokaryotic Translation David M. Bedwell, Ph.D. Department of Microbiology BBRB 432A Phone: 934-6593 E-mail: dbedwell@uab.edu Assigned Reading: Molecular Biology of the Cell, 5th Ed., Ch. 6, pp. 366-387. Optional Reading: Biochemistry, 3rd Ed., by Voet & Voet, Ch. 32, pp. 1285-1342.

Section 1: Reading the Genetic Code

The Central Dogma of Molecular Biology: DNA RNA Protein Translation\ noun\ a rendering from one language into another The fundamental problem: How do you get from one language based on 4 nucleotide code to another with a 20 amino acid code? Two Models were proposed: George Gamow: Proposed mechanism mediated by direct templating Francis Crick: Proposed mechanism utilizing adaptors

Crick’s Adaptor Hypothesis Crick’s Predictions Currently Known As: “…the RNA of the microsomal particles, regularly arranged, is the template” “…whatever went into the template in a specific way did so by forming hydrogen bonds” “…the amino acid is carried to the template by an adaptor...” “such adaptors…might contain nucleotides” “…a separate enzyme would be required to join each adaptor to its own amino acid…” “…the specificity required to distinguish between … isoleucine and valine would be provided by these enzymes” mRNA Codon-Anticodon Interactions Aminoacyl-tRNA tRNA Aminoacyl-tRNA Synthetase Editing by Aminoacyl-tRNA synthetases Crick, FHC. 1958. Symp. Soc. Exp. Biol. 12: 138-163.

The Standard Genetic Code Key Points: Multiple codons can encode the same amino acid (synonyms). Number of codons ranges from 1 to 6 for each amino acid. Some codons act for punctuation (start and stop codons). Figure 6-50 Molecular Biology of the Cell (© Garland Science 2008)

The Standard Genetic Code The genetic code: 64 codons Includes 1 initiation (start) and 3 termination (stop) codons Start Codon: AUG Stop Codons: UAA, UAG, UGA

The Reading Frame Problem How is the proper reading frame set? Start codon (AUG) sets the beginning of translation in the proper reading frame. Ribosome makes sure that the same reading frame is maintained until a stop codon terminates translation. Figure 6-51 Molecular Biology of the Cell (© Garland Science 2008)

tRNA Molecules Serve as Adaptors During Translation Figure 6-52 Molecular Biology of the Cell (© Garland Science 2008)

Achieving the Tertiary Structure of tRNA Molecules

Base-Pairing Between Codons and Anticodons Codon-anticodon pairing is anti-parallel. Base pairing between positions 1 and 2 use Watson-Crick pairing rules. Base pairing at position 3 (the wobble position) uses relaxed rules that allow more base pairing possibilities. Wobble allows some organisms to use as few as 32 tRNAs to translate entire genetic code. Figure 6-53 Molecular Biology of the Cell (© Garland Science 2008)

Section 2: Key Components of the Translational Machinery

Some Unusual Nucleotides Found in tRNAs Figure 6-55 Molecular Biology of the Cell (© Garland Science 2008)

Amino Acid Activation (tRNA Charging) Figure 6-56 Molecular Biology of the Cell (© Garland Science 2008)

Structure of the Aminoacyl-tRNA Linkage Figure 6-57 Molecular Biology of the Cell (© Garland Science 2008)

The Genetic Code is Translated by Two Adaptors That Work Sequentially Figure 6-58 Molecular Biology of the Cell (© Garland Science 2008)

Accuracy is Maintained by Hydrolytic Editing of AA-tRNA Synthetases Figure 6-59a Molecular Biology of the Cell (© Garland Science 2008)

Identity Elements in tRNAs Size of the yellow balls Are proportional to the fraction of 20 tRNA acceptor types for which the nucleoside is an observed determinant.

tRNA Recognition by its AA-tRNA Synthetase Figure 6-60 Molecular Biology of the Cell (© Garland Science 2008)

The Incorporation of an Amino Acid into a Growing Polypeptide Chain on the Ribosome Figure 6-61 Molecular Biology of the Cell (© Garland Science 2008)

Comparison of Prokaryotic and Eukaryotic Ribosomes Figure 6-63 Molecular Biology of the Cell (© Garland Science 2008)

The RNA Binding Sites in the Ribosome Figure 6-64 Molecular Biology of the Cell (© Garland Science 2008)

The Path of mRNA Through the Small Ribosomal Subunit Figure 6-65 Molecular Biology of the Cell (© Garland Science 2008)

Translation in Prokaryotes Initiates with N-Formylmethionine and tRNAfMet Shaded areas illustrate differences between initiator tRNA and other tRNAs

Section 3: The Translation Process Ribosome-Dependent Phases: Initiation Elongation Termination

Formation of the 30S Initiation Complex 30S pre-initiation complex

Formation of the 70S Initiation Complex

Elongation: Translating an mRNA Molecule Steps 1, 2: peptide bond formation Steps 3, 4: translocation Figure 6-66 Molecular Biology of the Cell (© Garland Science 2008)

Detailed (?) View of Prokaryotic Translation Elongation Factors: EF-Tu brings in each AA- tRNA. EF-G facilitates translocation to next codon. Figure 6-67 Molecular Biology of the Cell (© Garland Science 2008)

Translation Termination Release factors: RF-1: recognizes UAA, UAG codons RF-2: recognizes UAA, UGA codons RF-3: GTPase that facilitates RF-1 and RF-2 release after polypeptide release. Subsequent disassembly of post-termination complex mediated by two factors: Ribosome Recycling Factor (RRF) EF-G Figure 6-74 Molecular Biology of the Cell (© Garland Science 2008)

Effects of GTP Hydrolysis On EF-Tu Structural Dynamics

3D View of Translation Elongation

Dynamics of Ribosome Movement

Recognition of the First Base-Pair of a Correct Codon-Anticodon Pairing by Specific Residues of 16S rRNA Figure 6-68 Molecular Biology of the Cell (© Garland Science 2008)

Secondary Structure of Large Subunit rRNA in a Prokaryotic Ribosome Figure 6-69b Molecular Biology of the Cell (© Garland Science 2008)

Structure of Large Subunit rRNAs in a Prokaryotic Ribosome Figure 6-69a Molecular Biology of the Cell (© Garland Science 2008)

Location of Protein Components of the Large Subunit of a Prokaryotic Ribosome Figure 6-70 Molecular Biology of the Cell (© Garland Science 2008)

Structure of L15 Protein in the Large Subunit of a Prokaryotic Ribosome Figure 6-71 Molecular Biology of the Cell (© Garland Science 2008)

Structure of a Typical Prokaryotic mRNA Figure 6-73 Molecular Biology of the Cell (© Garland Science 2008)

Mechanism of Start Site Selection and Translational Control Base pairing between the Shine Dalgarno sequence and the 3´ end of 16S rRNA facilitates translation initiation. Consequently, the efficiency of translation initiation is determined by: 1) How well the S.D. sequence conforms to the consensus sequence that is complementary to the 3´ end of 16S rRNA. 2) The distance between the S.D. sequence and the start codon (a 7 base spacer is optimal).

Some Prokaryotic Translation Initiation Signals

Multiple Ribosomes Can Translate an mRNA Simultaneously in a Polysome Complex

Translational Recoding- Exceptions to the Basic Rules Figure 6-77 Molecular Biology of the Cell (© Garland Science 2008)

Binding Sites for Antibiotics on the Prokaryotic Ribosome Figure 6-79 Molecular Biology of the Cell (© Garland Science 2008)

Table 6-4 Molecular Biology of the Cell (© Garland Science 2008)

Puromycin is a Charged tRNA Analog

Rescue of a Prokaryotic Ribosome Stalled on a truncated mRNA Molecule Transfer-messenger RNA (abbreviated tmRNA) is a bacterial RNA molecule with dual tRNA-like and mRNA-like properties. The tmRNA forms a ribonucleoprotein complex (tmRNP) together with SmpB and EF-Tu. In trans-translation, tmRNA and its associated proteins bind to bacterial ribosomes which have stalled in the middle of protein synthesis, (e.g. at the end of an mRNA that has lost its stop codon). The tmRNA adds a proteolysis-inducing 11 AA tag on the unfinished polypeptide, recycles the stalled ribosome, and facilitates degradation of the aberrant mRNA. Figure 6-81 Molecular Biology of the Cell (© Garland Science 2008)

Trans-Translation Removes All Components of Stalled Translation Complexes tmRNA binds to SmpB and is aminoacylated by alanyl-tRNA synthetase (AlaRS). EF-Tu in the GTP state binds to alanyl-tmRNA, activating the complex for ribosome interaction (box 1). The alanyl-tmRNA/SmpB/EF-Tu complex recognizes ribosomes at the 3′end of an mRNA and enters the A-site as though it were a tRNA. The nascent polypeptide is transferred to tmRNA, and the tmRNA tag reading frame replaces the mRNA in the decoding center. The mRNA is rapidly degraded (box 2). Translation resumes, using tmRNA as a message, resulting in addition of the tmRNA-encoded peptide tag to the C terminus of the nascent polypeptide. Translation terminates at a stop codon in tmRNA, releasing the ribosomal subunits and the tagged protein. Multiple proteases recognize the tmRNA tag sequence and rapidly degrade the protein (box 3).

Co-Translational Protein Folding Figure 6-84 Molecular Biology of the Cell (© Garland Science 2008)

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