Protein Synthesis: Translation of the Genetic Message Chapter Twelve

Translating the Genetic Message

• Protein biosynthesis is a complex process requiring ribosomes, mRNA, tRNA, and

protein factors

• Several steps are involved • Before being incorporated into growing protein chain, a.a. must be activated by tRNA and

aminoacyl-tRNA synthetases

Salient features of the genetic code

– Triplet: a sequence of three bases (a codon) is needed to specify one amino acid – Nonoverlapping: no bases are shared between consecutive codons – Commaless: no intervening bases between codons – Degenerate: more than one triplet can code for the same amino acid; Leu, Ser, and Arg, for example, are each coded for by six triplets – Universal: the same in viruses, prokaryotes, and eukaryotes

The Genetic Code

• The ribosome moves along the mRNA three bases at a time rather than one or two at a time

The Genetic Code

• All 64 codons have assigned meanings –

61 code for amino acids

– 3 (UAA, UAG, and UGA) serve as termination

signals

– only Trp and Met have one codon each

The Genetic Code

• The third base is irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, and Arg •

Amino acids coded by 2, 3, or 4 triplets - the third letter of the codon - Gly, for example, is coded by GGA, GGG, GGC, and GGU

Wobble Base Pairing

• Some tRNAs bond to one codon exclusively, but many tRNAs can recognize more than one codon because of

variations in allowed patterns of hydrogen bonding

–

the variation is called “wobble”

–

wobble is in the first base of the anticodon

Base Pairing Combination in the Wobble Scheme

•

If there are 64 codons, how can there be less than 64 tRNA molecules?

The wobble hypothesis provides insight – in many cases, the degenerate codons for a given amino acid differ only in the third 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 misreading of the code; for example, if the Leu codon CUU were misread as 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 aminoacyl-tRNA synthetase Free energy of hydrolysis of ATP provides energy for bond formation

tRNA Tertiary Structure

• There are several recognition sites for various amino acids on the tRNA

Chain Initiation

• • • In all organisms, synthesis of polypeptide chain starts at the N-terminal end, and grows from N terminus to C-terminus Initiation requires: – tRNA fmet – initiation codon (AUG) of mRNA – 30S ribosomal subunit – – 50S ribosomal subunit initiation factors IF-1, IF-2, and IF-3 – GTP, Mg 2+ Forms the initiation complex

The Initiation Complex

Chain Initiation

– –

tRNA met

and tRNA

fmet

contain the triplet 3’-UAC-5’ Triplet base pairs with 5’-AUG-3’ in mRNA – 3’-UAC-5’ triplet on tRNA polypeptide synthesis

fmet

recognizes the AUG triplet (the start signal) when it occurs at the beginning of the mRNA sequence that directs – 3’-UAC-5’ triplet on tRNA triplet when it is found in an internal position in the mRNA sequence

met

recognizes the AUG

How does the ribosome know where to start translating

– Start signal is preceded by a Shine-Dalgarno

purine-rich leader segment, 5’-GGAGGU-3’

– Lies about 10 nucleotides upstream of the AUG start signal and acts as a ribosomal binding site

Chain Elongation

• • Uses three binding sites for tRNA present on the 50S subunit of the 70S ribosome: P (peptidyl) site, A (aminoacyl) site, E (exit) site.

Requires –

70S ribosome

–

codons of mRNA

–

aminoacyl-tRNAs

– elongation factors EF-Tu (Elongation factor temperature- unstable), EF-Ts (Elongation factor temperature-stable), and EF-G (Elongation factor-GTP) –

GTP, and Mg 2+

Chain Elongation

Why is EF-Tu so important in E.coli?

• • • Involved in translation fidelity tRNA and aminoacid are mismatched then EF Tu will not bind to t-RNA and will not deliver it to ribosome Binds activated tRNA too well and does not release it from ribosome

Elongation Steps

• • • •

Step 1

– an aminoacyl-tRNA is bound to the A site – the P 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 peptide bond is formed, the P site is uncharged

Step 4

– the uncharged tRNA is released – the peptidyl-tRNA is translocated to the P site – – EF-G and GTP are required the next aminoacyl-tRNA occupies the empty A site

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 – It is possible to have several molecules of RNA polymerase bound to a single DNA gene, each in a different stage of transcription – It is also possible to have several ribosomes bound to a single mRNA, each in a different stage of translation – – Polysome: mRNA bound to several ribosomes Coupled translation: 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 simultaneously • Each ribosome produces a copy of the polypeptide chain specified by the mRNA • When protein has been completed, the ribosome dissociates into subunits that are used again in protein synthesis

Simultaneous Protein Synthesis on Polysomes

•

Eukaryotic Translation

Chain Initiation: • the most different from process in prokaryotes • 13 more initiation factors are given the designation eIF (eukaryotic initiation factor) (Table 12.4)

Eukaryotic Translation

•

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

– stop codons are the same: UAG, UAA, and UGA – only one release factor that binds to all three stop codons

Posttranslational Modification

• Newly synthesized polypeptides are frequently modified before they reach their final form where they exhibit biological activity – N-formylmethionine in prokaryotes is cleaved – leader sequences 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 disulfide 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

• Degradative pathways are restricted to – subcellular organelles such as lysosomes – macromolecular structures called proteosomes

Protein Degradation

• In eukaryotes,

ubiquitinylation

(becoming bonded to ubiquitin) targets a protein for destruction – those with an N-terminus of Met, Ser, Ala, Thr, Val, Gly, and Cys are resistant – those with an N-terminus of Arg, Lys, His, Phe, Tyr, Trp, Leu, Asn, Gln, Asp, Glu have short half-lives

Acidic N-termini Induced Protein Degradation

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•

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