Transcript Prokaryotic Translation - Department of Microbiology

Prokaryotic
Translation
David M. Bedwell, Ph.D.
Department of Microbiology
BBRB 432A
Phone: 934-6593
E-mail: [email protected]
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
– “…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”
Crick, FHC. 1958. Symp. Soc. Exp. Biol. 12: 138-163.
Currently Known As:
mRNA
Codon-Anticodon
Interactions
Aminoacyl-tRNA
tRNA
Aminoacyl-tRNA
Synthetase
Editing by Aminoacyl-tRNA
synthetases
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?
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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 antiparallel.
• 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 AAtRNA.
• 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 CodonAnticodon 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
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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
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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)
Ramakrishnan Movie
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