Transcript fa458c46b7c1dda

Chapter 22
Gene Expression:
II. Protein
Synthesis and
Sorting
Lectures by
Kathleen Fitzpatrick
Simon Fraser University
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Gene Expression: II. Protein Synthesis
• For some genes, the RNA is the final product
• But for most genes, the ultimate product is protein
• mRNAs encode instructions for translation,
the assembling amino acids into a polypeptide
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Translation: The Cast of Characters
• Ribosomes carry out the polypeptide synthesis
• tRNA molecules transport the amino acids
• Aminoacyl-tRNA synthetases attach amino acids to
their appropriate tRNA molecules
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The cast of characters
• mRNA molecules encode the amino acid
sequence information
• Protein factors facilitate the steps of translation
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The Ribosome Carries Out Polypeptide
Synthesis
• Ribosomes: particles made of rRNA and protein
• In eukaryotes: found free in the cytoplasm, bound to
ER and the outer nuclear envelope
• Ribosomes are built from dissociable subunits, the
large and small subunits
• In prokaryotes, the ribosomes are smaller
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Figure 22-1B
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Table 22-1
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Ribosomes, machines in polypeptide
synthesis
• rRNA performs the key functions of ribosomes
• Ribosomes have four important sites: the mRNA
binding site, the A (aminoacyl) site, the
P (peptidyl) site, and an E (exit) site
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Figure 22-2
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Transfer RNA Molecules Bring Amino
Acids to the Ribosome
• A tRNA molecule is an adaptor that binds both a
specific amino acid and the mRNA sequences that
specify the amino acid
• Each tRNA is linked to its amino acid by ester bond
• tRNAs are named for the amino acids attached to
them, e.g., tRNAAla for alanine
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Figure 22-3A
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Figure 22-3B
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tRNAs
• tRNAs attached to an amino acid are said to be
aminoacyl tRNAs—the tRNA is called charged,
whereas the amino acid is called activated
• Each tRNA recognizes codons in mRNA due to their
complementarity to the anticodon in the tRNA
• Some tRNAs recognize more than one codon
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The wobble hypothesis
• mRNA and tRNA line up on the ribosome in a way
that permits flexibility or wobble in the pairing
between the third base of the codon and the
corresponding base of the anticodon
• This is the wobble hypothesis, which allows for
some unexpected base pairing
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Figure 22-4
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Inosine
• The nucleotide inosine is able to pair with U, C, or
A and is often found in the wobble position of the
anticodon
• This allows for several codons to specify one
amino acid
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Aminoacyl-tRNA Synthetases Link
Amino Acids to the Correct
Transfer RNAs
• Cells typically have 20 different aminoacyl-tRNA
synthetases to attach each amino acid to the
appropriate tRNA
• There is one aminoacyl-tRNA synthetase for each
amino acid
• Cells with nontraditional amino acids have special
tRNAs and aminoacyl-tRNA synthetases for these
amino acids, too
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Aminoacyl-tRNA synthesis
• Aminoacyl-tRNA synthetases catalyze the
attachment of amino acids to the tRNAs via an
ester bond, using ATP hydrolysis
•
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Aminoacyl-tRNA synthesis
• Both the anticodon and the 3 end of the tRNA are
needed to specify the correct amino acid
• After addition of an amino acid the synthetases
proofread the final product to ensure the correct
amino acid was added
• It is the tRNA that then recognizes the appropriate
codon in mRNA
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Figure 22-5, Steps 1, 2
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Figure 22-5, Steps 3, 4
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Messenger RNA Brings Polypeptide
Coding Information to the Ribosome
• The sequence of codons in mRNA directs the order
of amino acids in the polypeptide
• mRNA is exported to the cytoplasm via binding to
proteins that contain nuclear export signals (NES)
• An untranslated sequence at the 5 end of the
message precedes the start codon, the first to be
translated (usually AUG)
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Coding information
• There is an untranslated region at the 3 end of the
mRNA that follows the stop codon, which signals
the end of translation
• The stop codon may be UAG, UAA, or UGA
• 5 and 3 untranslated regions vary in length and
are essential for mRNA function
• mRNAs also have a 5 cap and 3 poly(A) tail
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Figure 22-6
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Eukaryotic mRNAs are monocistronic
• Most mRNAs in eukaryotes are monocistronic,
meaning they encode just one polypeptide
• In bacteria and archaea, some are polycistronic,
encoding several polypeptides with related functions
• Polycistronic transcription units are called operons
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Protein Factors Are Required for
the Initiation, Elongation, and
Termination of Polypeptide Chains
• Translation is an ordered, stepwise process that
begins at the N-terminus of the polypeptide and
adds amino acids to the growing chain until the Cterminus is reached
• The mRNA is read in the 5 to 3 direction
• Translation is divided into three stages: initiation
(1), elongation (2), and termination (3)
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Figure 22-7
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The Initiation of Translation Requires Initiation
Factors, Ribosomal Subunits, mRNA, and
Initiator tRNA Eukaryotic Initiation
• The start codon in eukaryotes and archaea
specifies methionine
• The initiation factors are called eIFs; there are
about a dozen of these
• eIF2 (with GTP attached) binds to the initiator
tRNAMet before the tRNA then binds the small
ribosomal subunit
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Eukaryotic initiation (continued)
• After binding the mRNA, the small ribosomal subunit
(including the initiator tRNA) scans along the
transcript and begins translation at the first AUG
• Nucleotides to either side of the start codon are
involved in the recognition; e.g., a common start
sequence is ACCAUGG, called a Kozak sequence
• After the initiator tRNA is base-paired with the start
codon the large subunit joins the complex, facilitated
by GTP hydrolysis
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Chain Elongation Involves Sequential
Cycles of Aminoacyl tRNA Binding,
Peptide Bond Formation, and
Translocation
• Once initiation has been completed a polypeptide
chain is synthesized
• Amino acids are added in sequence to the growing
chain (elongation)
• Elongation involves a repetitive cycle of three steps
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Binding of Aminoacyl tRNA
• As elongation begins, the start codon is located at
the P site and the next codon is at the A site
• Elongation begins as a tRNA with an anticodon
complementary to the second codon binds the A
site (1)
• This requires two elongation factors, EF-Tu and
EF-Ts, and GTP hydrolysis
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Figure 22-10
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Binding of aminoacyl tRNA (continued)
• Elongation factors don’t recognize particular
anticodons, so all types are brought to the A site
• Only those with an anticodon complementary to the
codon stay at the A site long enough for GTP
hydrolysis to take place
• The final error rate in translation is at most 1/10,000
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Peptide Bond Formation
• Once the aminoacyl tRNA is bound to the A site, a
peptide bond forms between the amino group of the
amino acid at the A site and the carboxyl group of
the amino acid at the P site
• The growing peptide chain is transferred to the
tRNA at the A site (2)
• No ATP or GTP hydrolysis is required for this step
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rRNA catalyzes peptide bond formation
• It was thought that the protein peptidyl
transferase catalyzed peptide bond formation
• However, Noller and colleagues showed that rRNA
contains the catalytic activity
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Translocation
• After the peptide bond forms, the mRNA advances to bring
the next codon into the proper position
• During this translocation, the peptidyl tRNA moves from
the A to the P site, and the empty tRNA moves to the E site
• Hydrolysis of GTP bound to EF-G triggers a conformational
change that completes these movements (3)
• Once the next mRNA codon reaches the A site,
the ribosome is now set to receive the next aminoacyl tRNA
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Termination of Polypeptide Synthesis
Is Triggered by Release Factors That
Recognize Stop Codons
• Codons are read on the mRNA one after the other,
until a stop codon arrives at the A site
• Stop codons are recognized by protein release
factors, rather than tRNAs
• Once release factors bind to the stop codons,
translation is terminated through release of the
completed polypeptide
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Figure 22-11
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Polypeptide Folding Is Facilitated by
Molecular Chaperones
• Proteins must fold into their correct threedimensional shapes before they can function
• Protein folding is usually facilitated by proteins
called molecular chaperones; often several are
required, acting in sequence
• Chaperones bind polypeptide chains during the
early stages of folding
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Molecular chaperones
• If folding goes awry, chaperones can sometimes
rescue the proteins and fold them properly
• Alternatively, improperly folded proteins may be
destroyed
• Some kinds of incorrectly folded proteins bind to
each other and form insoluble aggregates within and
between cells
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Protein Synthesis Typically Utilizes
a Substantial Fraction of a Cell’s
Energy Budget
• Polypeptide elongation involves hydrolysis of at
least four high-energy phosphoanhydride bonds
• Assuming each bond has a G°of 7.3 kcal/mol,
they represent a free energy input of 29.2 kcal/mol
• Additional GTPs are used during formation of the
initiation complex, the binding of incorrect
aminoacyl tRNAs, and termination
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A Summary of Translation
• Translation converts information in mRNAs into a
chain of amino acids linked by peptide bonds
• Most messages are read by many ribosomes
simultaneously; a cluster of such ribosomes
attached to the same mRNA is called a
polyribosome
• RNA molecules play important roles in translation;
mRNA, tRNA, rRNA
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Mutations and Translation
• mRNAs may contain mutant codons that cause
errors in the polypeptide chain synthesized
• Most codon mutations alter a single amino acid and
some (in the third base of a codon) don’t alter the
amino acid at all
• Mutations that add or remove stop codons or alter
the reading frame can severely disrupt translation
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Suppressor tRNA Overcomes the
Effects of Some Mutations
• Mutations that convert amino acid-coding codons
into stop codons, called nonsense mutations,
typically lead to incomplete, nonfunctional
polypeptides
• These mutations are often lethal, but can
sometimes be overcome by an independent
mutation affecting a tRNA gene
• This is called a suppressor tRNA
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Figure 22-12A, B
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Suppressor tRNAs
• Suppressor tRNAs recognize stop codons and
insert amino acids, suppressing nonsense
mutations
• Highly efficient suppressor tRNAs might lead to the
production of many abnormal amino acids
• However, at the 3 ends of mRNAs, release factors
bind stop codons more efficiently than suppressor
tRNAs; this limits the effect of the suppressor to
internal locations on the mRNA
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Figure 22-12B, C
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Nonsense-Mediated Decay and
Nonstop Decay Promote the
Destruction of Defective mRNAs
• Without a suppressor tRNA, a nonsense mutation
will cause premature termination of translation and
an incomplete polypeptide chain
• Eukaryotic cells use nonsense-mediated decay to
destroy mRNAs containing premature stop codons
• In mammals, the exon junction complex (EJC) is
used to detect premature stop codons
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The EJC and nonsense mutations
• A multiprotein EJC is deposited wherever an intron
is removed from pre-mRNA, so each spliced mRNA
has at least one complex bound to it
• If an mRNA contains a stop codon prior to the final
EJC, translation is terminated
• EJCs still associated with the tRNA target it for
degradation
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The fate of mRNAs with no stop codon
• In eukaryotes, translation is stalled when a
ribosome reaches the end of a transcript that lacks
a stop codon
• An RNA degrading enzyme binds the empty A site
of the ribosome and degrades the defective mRNA
via nonstop decay
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Posttranslational Processing
• After polypeptide chains are synthesized, they often must
undergo posttranslational modification before they can
perform their functions
• In eukaryotes, the methionine at the N-terminus is released
•
Sometimes whole blocks of amino acids are removed from
the polypeptide, for instance certain enzymes synthesized
as inactive precursors, these are activated by removal of
sequences from one end of the protein
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Posttranslational processing (continued)
• Other common processing events include chemical
modification of amino acids—methylation,
phosphorylation, acetylation
• Some proteins undergo a rare process called
protein splicing
• Similar to RNA splicing, protein sequences called
inteins are removed and the remaining sequences
called exeins are spliced together
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