Chapter 6. Gene Expression: Translation

Transcription

1 Chapter 6 Gene Expression: Translation

2 Translation Nucleotides specify the amino-acid sequence in proteins with four different nucleotides. A, C, G and U. A three letter code (using three of the four nucleotides) generates 64 possible combinations or CODONS. These 64 combinations are more than enough to codify for the 20 amino acids that forms proteins.

3 Evidence for a triplet code came from experiments performed in the early 1960 s by Crick, Barner, Brennet and Watts-Tobin in bacteriophage T4. The exact relationship of the 64 codons to the 20 Amino Acids was determined by Nirenberg and Khorana in Based on Nirenberg and Khorana s work, we know that by convention, a codon is written as it appears in mrna, reading in the 5 to 3 direction.

4 Characteristics of the Genetic Code a. It is a triplet code. Each three-nucleotide codon in the mrna specifies one amino in the polypeptide. b. It is comma free. The mrna is read continuously, three bases at a time, without skipping any bases. c. It is non-overlapping. Each nucleotide is part of only one codon, and is read only once during translation.

5 d. It is almost universal. In nearly all organisms studied, most codons have the same amino acid meaning. e. It is degenerate. Of 20 amino acids, 18 are encoded by more than one codon. Met (AUG) and Trp (UGG) are the exceptions; all other amino acids correspond to a set of two or more codons. Codon sets often show a pattern in their sequences; variation at the third position is most common. f. The code has start and stop signals. AUG is the usual start signal for protein synthesis and defines the open reading frame. Stop signals are codons with no corresponding trna, the nonsense or chain-terminating codons. There are generally three stop codons: UAG (amber), UAA (ochre), and UGA (opal).

6 g. If each anticodon had to be a perfect match to each codon, we would expect to find 61 types of trna, but the actual number is about 45. This is because the anticodons of some trnas recognize more than one codon. This is possible because the rules for base pairing between the third base of the codon and anticodon are relaxed (called wobble). At the wobble position, U on the anticodon can bind with A or G in the third position of a codon. Some trna anticodons include a modified form of adenine, inosine, which can hydrogen bond with U, C, or A on the codon.

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8 Translation: The Process of Protein Synthesis Protein synthesis take places in the ribosomes where the genetic message encoded in mrna is decoded and translated. The mrna is translated 5 -to-3, and the polypeptide is made in the N-terminal- to- C-terminal direction. Amino acids are brought to the ribosomes bound to trnas and the amino acids are inserted in the proper sequence due to: The specific binding of each amino acid to its trna. The specific base-pairing between the mrna codon and complementary trna anticodon.

9 The trna All trnas can be shown in a cloverleaf structure, with complementary base pairing between regions to form four stems and loops (Figure 5.20). Loop II contains the anticodon used to recognize mrna codons during translation. Folded trnas resemble an upside-down down L. L.

10 Details 1. How does the trna add an amino acid? The amino acid is attached to the trna by the enzyme aminoacyl trna synthetase. The process is called amino acetylation and the trna with an amino acid is called charged trna.

11 Details 2. How does the mrna recognizes the anticodon? The mrna recognizes the anticodon but not the amino acid carried by the trna.

12 Translation 1. Protein synthesis is similar in prokaryotes and eukaryotes. Some significant differences do occur and we will mention as we go. 2. In both it is divided into three stages: a. Initiation. b. Elongation. c. Termination. In prokaryotes initiation of translation requires: A mrna, a ribosome, a specific initiator trna, initiation factors, GTP for energy and Mg2+ (magnesium ions).

13 Initiation In prokaryotes the first step is the boinding of the small ribosome subunit (30s) to the mrna region where the AUG (start codon) is located. 1. The 30S ribosomal subunit binds to IF1, IF2, IF3, GTP and Mg ions.

14 The mrna has a sequence upstream (5 ) of the start codon AUG (about 8-12 nt) known as the ribosome binding site or RBS. It is a purine (AG) rich area. The RBS is also known as the Shine-Dalgano sequence.

15 The recognition of the complementary base pairs between the RBS of the mrna and the 16S ribosomal RNA of the small ribosome subunit allows the ribosome to locate the precise location for the initiation of protein synthesis.

16 Next, the initiator trna binds the AUG to which the 30S subunit is bound. AUG universally encodes methionine. Newly made proteins begin with Met, which is often subsequently removed. In prokaryotes, initiator methionine is formylmethionine (fmet). It is carried by a specific trna (with the anticodon 5 - CAU-3 ). It is a methionine with a formil group. The rests of methionines in the chain are added by normal met trna.

17 When fmet-trna binds to the start codon of the 30S-mRNA complex, IF3 is released and the 50S ribosomal subunit binds the complex. GTP is hydrolyzed, and IF1 and IF2 are released. The result is a 70S initiation complex consisting of: a. mrna. b. 70S ribosome (30S and 50S subunits) with a vacant A site. c. fmet-trna in the ribosome s P site.

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19 The main differences in eukaryotic translation are: a. Initiator methionine is not modified. As in prokaryotes, it is attached to a special trna. b. Ribosome binding involves the 5 cap, rather than a Shine-Delgarno sequence and a large group of initiator factors. c. The eukaryotic mrna s 3 polya tail also plays a role in initiation. The PolyA binding protein (PABP) that is bound to the polya tail binds to the initiator factor bound to the cap, thus loping the 3 of the mrna close to the 5 end. The PoliA tail stimulates the inititation of translation.

20 Elongation of the Polypeptide Chain Elongation of the amino acid chain has three steps (Figure 6.13): a. Binding of aminoacyl-trna to the ribosome. b. Formation of a peptide bond. c. Translocation of the ribosome to the next codon.

21 REVIEW Each ribosome has a binding site for mrna and three binding sites for trna molecules. The P site holds the trna carrying the growing polypeptide chain. The A site carries the trna with the next amino acid. Discharged trnas leave the ribosome at the E site.

22 Binding of Aminoacyl-tRNA At the start of elongation the anticodon of fmet is bonded the AUG initiation codon (hydrogen bonds) in the P site. The next codon in th mrna is the A site. The EF-Tu is called protein elongation factor. The next charged trna approaches the ribosome bound to EF-Tu-GTP. When the charged trna hydrogen bonds with the codon in the ribosome s A site, hydrolysis of GTP releases EF-Tu-GDP.

23 EF-Tu is recycled with assistance from EF-Ts, which removes the GDP and replaces it with GTP, preparing EF-Tu-GTP to escort another aminoacyl trna to the ribosome.

24 Peptide Bond Formation 1. The two aminoacyl-trnas are positioned by the ribosome for peptide bond formation, which occurs in two steps (Figure 6.14): a. In the P site, the bond between the amino acid and its trna is cleaved. b. Peptidyl transferase forms a peptide bond between the now-free amino acid in the P site and the amino acid attached to the trna in the A site. c. The trna in the A site now has the growing polypeptide chain attached to it.

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26 Translocation 1. The ribosome now advances one codon along the mrna. EF-G (elongation factor G ) is used in translocation in prokaryotes. EF-G- GTP binds the ribosome, GTP is hydrolyzed, and the ribosome moves one codon while the uncharged trna leaves the P site. (Eukaryotes use a similar process, with a factor called eef-2.)

27 2. During translocation the peptidyl-trna remains attached to its codon, but is transferred from the ribosomal A site to the P site by an unknown mechanism. 3. Release of the uncharged trna involves the 50S ribosomal E (for Exit) site. Binding of a charged trna in the A site is blocked until the spent trna is released from the E site.

28 4. The vacant A site now contains a new codon, and an aminoacyl-trna with the correct anticodon can enter and bind. The process repeats until a stop codon is reached. 5. Elongation and translocation are similar in eukaryotes, except for differences in number and type of elongation factors and the exact sequence of events. 6. In both prokaryotes and eukaryotes, simultaneous translation occurs: New ribosomes may initiate as soon as the previous ribosome has moved away from the initiation site, creating a polyribosome (polysome); an average mrna might have 8 10 ribosomes (Figure 6.15).

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30 Termination Termination is signaled by a stop codon (UAA,UAG,UGA), which has no corresponding trna (Figure 6.16). Release factors (RF) assist the ribosome in recognizing the stop codon and terminating translation. In E. coli (RF1, RF2 and RF3). In eukaryotes, erf is the only termination factor, recognizing all three stop codons and stimulating termination.

31 Sequence of termination events 1. Stop codon is encountered and there is not amynoacyltrna that corresponds with a stop codon. 2. A release factor binds to the stop codon. 3. The polypeptide chain is realease from the trna in the A site. 4. All the components separate. 5. Methionine or fmet are cleaved and removed.

32 Protein sorting How does the protein know where to go? Is it a protein that is going to go to an organelle or is it going to be secreted? In eukaryotes, proteins synthesized on the rough ER (endoplasmic reticulum) are glycosylated (signaled) and then transported in vesicles to the Golgi apparatus. The Golgi sorts proteins based on their signals, and sends them to their destinations.

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