Transcript Document

Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
Chapter Twelve
Protein Synthesis:
Translation of the Genetic Message
Paul D. Adams • University of Arkansas
Translating the Genetic Message
• Protein biosynthesis is a
complex process
requiring
_________________,
_________________, &
_________________
• Several steps are
involved
• Before being
incorporated into
growing protein chain,
a.a. must be _________
by tRNA and
___________________
The Genetic Code
• Salient features of the genetic code
• ___________: a sequence of three bases (a codon) is
needed to specify one amino acid
• _________________: no bases are shared between
consecutive codons
• ___________: no intervening bases between codons
• ____________________: more than one triplet can
code for the same amino acid; Leu, Ser, and Arg, for
example, are each coded for by six triplets
• _______________: the same in viruses, prokaryotes,
and eukaryotes; the only exceptions are some codons
in mitochondria
The Genetic Code (Cont’d)
The __________ moves
along the mRNA ______
bases at a time rather
than ________ at a time
Theoretically possible
genetic codes are shown
in figure 12.2
The Genetic Code (Cont’d)
• All 64 codons have assigned meanings
• 61 code for ____________ _____________
• 3 (UAA, UAG, and UGA) serve as _____________
_____________ signals
• only ______ and _______ have one codon each
• the ______ ______ base is irrelevant for Leu, Val,
Ser, Pro, Thr, Ala, Gly, and Arg
• the second base is important for the ______ of amino
acid; for example, if the second base is U, the amino
acids coded for are hydrophobic
• for the 15 amino acids coded for by 2, 3, or 4 triplets,
only the _______ letter of the codon varies. For
example, Gly is coded for by GGA, GGG, GGC, and
GGU
The Genetic Code (Cont’d)
The Genetic Code (Cont’d)
• Assignments of triplets in genetic code based on
_________________________________________
• ______ mRNA: if mRNA is polyU, polyPhe is formed;
if mRNA is poly --ACACACACACACACACACACA--,
poly(Thr-His) is formed
• _____________: aminoacyl-tRNAs bind to ribosomes
in the presence of trinucleotides
• synthesize ________________ by chemical means
• carry out a ________________ for each type of
trinucleotide
• _______________-tRNAs are tested for their ability
to bind in the presence of a given trinucleotide
The Filter-Binding Assay
Wobble Base Pairing
Some tRNAs bond to one codon exclusively, but
many tRNAs can recognize more than one codon
because of variations in ______________________
• the variation is called “wobble”
• wobble is in the _________ base of the _________
Base Pairing…Wobble Scheme
Wobble Base Pairing
Wobble Base Pairing Hypothesis
• The wobble hypothesis provides insight into some
aspects of the _________________ of the code
• in many cases, the degenerate codons for a given
amino acid differ only in the ______ base; therefore
fewer different tRNAs are needed because a given
tRNA can base-pair with several codons
• the existence of wobble minimizes the damage that
can be caused by a _______________ of the code;
for example, if the Leu codon CUU were misread
CUC or CUA or CUG during transcription of mRNA,
the codon would still be translated as Leu during
protein synthesis
Amino Acid Activation
• Amino acid activation
and formation of the
aminoacyl-tRNA take
place in two separate
steps
• Both catalyzed by
amionacyl-tRNA
_________________
• Free energy of
hydrolysis of _______
provides energy for
__________________
Amino Acid Activation (Cont’d)
This two-stage reaction allows selectivity at two levels
• ______________: the aminoacyl-AMP remains bound
to the enzyme and binding of the correct amino acid is
verified by an editing site in the tRNA synthetase
• ________: there are specific binding sites on tRNAs
that are recognized by aminoacyl-tRNA synthetases.
tRNA Tertiary Structure
Recognition sites for various amino acids on the tRNA
Chain Initiation
• In all organisms, synthesis of polypeptide chain
starts at the ____________ end, and grows from
_________________ to ________________
• Initiation requires:
•
•
•
•
•
•
tRNAfmet
initiation codon (AUG) of mRNA
30S ribosomal subunit
50S ribosomal subunit
initiation factors IF-1, IF-2, and IF-3
GTP, Mg2+
• Forms the _______________ _______________
The
Initiation
Complex
Chain Initiation
• tRNAmet and tRNAfmet contain the triplet 3’-_______-5’
• Triplet base pairs with 5’-AUG-3’ in mRNA
• 3’-UAC-5’ triplet on tRNAfmet recognizes the AUG
triplet (start signal) when it is at the ____________ of
the mRNA sequence that directs polypeptide
synthesis
• 3’-UAC-5’ triplet on tRNAmet recognizes the AUG
triplet when it is found in an _________________
_________________ in the mRNA sequence
• Start signal is preceded by a Shine-Dalgarno purinerich leader segment, 5’-GGAGGU-3’, which usually
lies about ___ nucleotides _______________ of the
AUG start signal and acts as a ________ binding site
Chain Elongation
• Uses 3 binding sites for tRNA present on the 50S
subunit of the 70S ribosome:
P (___________) site, A (________) site, E (______) site
• Requires
•
•
•
•
•
70S ribosome
codons of mRNA
aminoacyl-tRNAs
GTP, and Mg2+
elongation factors:
• EF-Tu (Elongation factor temperature-unstable),
• EF-Ts (Elongation factor temperature-stable), and
• EF-G (Elongation factor-GTP)
Shine-Dalgarno Sequence Recognized by
E. Coli Ribosomes
Elongation Steps
• Step 1
• an aminoacyl-tRNA is bound to the ___ site
• the ___ site is already occupied
• 2nd amino acid bound to 70S initiation complex. Defined by the
mRNA
•
Step 2
• EF-Tu is released in a reaction requiring EF-Ts
• Step 3
• the _______ _______is formed, the P site is uncharged
• Step 4
•
•
•
•
the uncharged tRNA is released
the _____________-tRNA is translocated to the P site
EF-G and GTP are required
the next aminoacyl-tRNA occupies the _________ ___ site
Chain Elongation
Chain Termination
• Chain termination requires
• stop codons (UAA, UAG, or UGA) of mRNA
• RF-1 (Release factor-1) which binds to UAA and
UAG or RF-2 (Release factor-2) which binds to UAA
and UGA
• RF-3 which does not bind to any termination codon,
but facilitates the binding of RF-1 and RF-2
• GTP which is bound to RF-3
• The entire complex dissociates, setting free the
completed polypeptide, the release factors, tRNA,
mRNA, and the 30S and 50S ribosomal subunits
Chain
Termination
Components of Protein Synthesis
Protein Synthesis
• In prokaryotes, translation begins very soon after
mRNA transcription
• Several molecules of RNA polymerase may be bound
to a single DNA gene, each in a different stage of
transcription
• Several ribosomes may be bound to a single mRNA,
each in a different stage of translation
• _____________: mRNA bound to several ribosomes
• _________________ _________________ : the
process in which a prokaryotic gene is being
simultaneously transcribed and translated
Simultaneous Protein Synthesis on Polysomes
• A single mRNA molecule is translated by several
ribosomes _________________
• Each ribosome produces a copy of the polypeptide
chain specified by the mRNA
• When protein has been completed, the ribosome
____________________________ that are used
again in protein synthesis
Simultaneous Protein Synthesis on Polysomes (Cont’d)
Eukaryotic Translation
• Eukaryotic Chain Initiation:
• the most different from process in prokaryotes
• 13 more _________ __________are given the designation
eIF (eukaryotic initiation factor) (Table 12.4)
Eukaryotic Translation (Cont’d)
• Chain elongation
• uses the same mechanism of peptidyl transferase and
ribosome translocation as prokaryotes
• there is no E site on eukaryotic ribosomes,
only A and P sites
• there are two elongation factors, eEF-1 and eEF-2
• eEF2 is the counterpart to EF-G, which causes
translocation
• Chain termination
• ______ codons are the same: UAG, UAA, and UGA
• only one _________ __________ binds to all three
_____________ _____________
Posttranslational Modification
• Newly synthesized polypeptides are frequently ___________
before they reach their final form where they exhibit
biological activity
• N-formylmethionine in prokaryotes is _______________
• specific bonds in precursors are cleaved, as for example,
preproinsulin to proinsulin to insulin
• ___________ _________are removed by specific proteases of
the endoplasmic reticulum; the Golgi apparatus then directs the
finished protein to its final destination
• factors such as heme groups may be attached
• ___________ bonds may be formed
• amino acids may be modified, as for example, conversion of
proline to hydroxyproline
• other covalent modifications; e.g., addition of carbohydrates
Examples of Posttranslational Modification
Protein Degradation
• Proteins are in a _________ state and are often
turned over
• Degradative pathways are restricted to
• subcellular organelles such as lysosomes
• macromolecular structures called proteosomes
• In eukaryotes, ____________ (becoming bonded to
_______________) targets a protein for destruction
• protein must have an N-terminus
• those with an N-terminus of Met, Ser, Ala, Thr, Val,
Gly, and Cys are ________________
• those with an N-terminus of Arg, Lys, His, Phe, Tyr,
Trp, Leu, Asn, Gln, Asp, Glu have ______ half-lives
Ubiquitin-Proteosome Degradation
Acidic N-termini Induced Protein
Degradation