Paul D. Adams • University of Arkansas

Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Chapter TwelveProtein Synthesis:

Translation of the Genetic Message

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: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 purine-rich 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