Protein Synthesis: Translation

Translation Translation: Assembly of polypeptides on a ribosome • Living cells devote more energy to the synthesis of proteins than to any other aspect of metabolism. • About a third of the dry mass of a cell consists of molecules that directly participate in protein synthesis • This reflects the importance of protein synthesis to the existence of the organism. 1 /35 Translation: An Overview • Ribosomes translate the genetic message of mRNA into proteins. • The mRNA is translated 5’ ! 3’ producing a corresponding Nterminal ! C-terminal polypeptide. • Amino acids bound to tRNAs are inserted in the proper sequence due to: – Specific binding of each amino acid to its tRNA. – Specific base-pairing between the mRNA codon and tRNA anticodon. 2 Components of Translation • mRNA: – Eukaryotes: made in the nucleus, transported to the cytoplasm. – Prokaryotes: transcription and translation occur concurrently. • tRNA: Adaptor molecules that mediate the transfer of information from nucleic acids to protein • Ribosomes: manufacturing units of a cell; located in the cytoplasm. Contain ribosomal RNA and proteins. • Enzymes: required for the attachment of amino acids to the correct tRNA molecule, and for peptide bond formation between amino acids. • Proteins: soluble factors necessary for proper initiation, elongation and termination of translation. 3 tRNA • small single stranded RNA molecules of 70-95 nucleotides in length, – about 4S (Svedberg units) in size. • In addition to A, G, C and U, tRNAs have modified bases produced by chemical alteration of the 4 primary nucleotides. • Each tRNA molecule is a clover leaf structure, which looks like an L-shape in three dimensions. • At the base of the L, three nucleotides form the anti-codon. • The sequence of the anti-codon dictates the amino acid that binds to it. – The anti-codon sequence is complementary to the codon for that amino acid. – For example: • GCA is a codon for alanine: the anticodon then is CGU, but in the 3’ to 5’ direction. • The amino acid is carried at the 3’ hydroxyl end of the tRNA molecule. 4 /35 tRNA • Tertiary structure • Amino acids must be attached to be functional – Enzymatic reaction – Need ATP – Aminoacyl tRNA synthase 5 /35 tRNA • Recognition of codon is important • Are there tRNAs for every codon? – So are there 61 tRNA’s? • No actually about 40 • “Wobble” in third position of anticodon • One anticodon can recognize several codons… • What are the wobble rules? 6 /35 Characteristics of the Genetic Code: The Wobble Hypothesis • Wobble occurs in the anticodon. – The third base in the codon is able to base-pair less specifically – because it is less constrained three-dimensionally. • allows a tRNA anticodon to recognize up to three different codons (Figure 14.8 and Table 14.1). – Does not obey complementary base pairing in certain cases. – YIKES! 7 Fig. 6.9 Example of base-pairing wobble 8 Some tRNAs recognize more than one codon for amino acids they carry 9 Enzymes • Aminoacyl-tRNA synthetases catalyze the attachment of a tRNA molecule to its respective amino acid. – There is at least one amino acyl tRNA synthetase for each amino acid. – The attachment of the amino acid activates/ charges the tRNA molecule. – The attachment of the aminoacid is at its carboxyl terminal. (NH2-CH2-CO-3’tRNA5’) • Peptidyl Transferase: – catalyzes the sequential transfer of amino acids to the growing chain. – Forms the peptide bonds between amino acids 12 /35 Ribosomes: Functions • They are the sites of polypeptide synthesis • They recognize features that signal the start of translation • They ensure the accurate interpretation of the genetic code by stabilizing the interaction between tRNA and the mRNA. • They supply the enzymatic activity that covalently links the amino acids in the polypeptide chain. • They facilitate the linear reading of the genetic code by sliding along the mRNA molecule. 13 /35 Ribosomes: Components • two subunits: large and small. – Prokaryotes: 50S + 30S = 70S – eukaryotes: 60S + 40S = 80S. • Prokaryotes: overall smaller – large subunit contains one rRNAs and ~31 different proteins. – small subunit contains two rRNAs and 21 different proteins. • Eukaryotes: overall bigger – large subunit contains three rRNAs and 45 proteins. – small subunit consists of one rRNAs and 33 different proteins. 14 /35 Ribosomes: Synthesis • In eukaryotes, rRNA synthesis and ribosome assembly takes place in the nucleolus. • Before translation begins, the two ribosomal subunits exist as separate entities in the cytoplasm. • Soon after the start of translation, they come together. 15 /35 Ribosomes: Role in translation • The small subunit is the one that initially binds to the mRNA. • The larger subunit provides the enzyme activity: •Peptidyl transferase, •catalyzes formation of peptide bonds joining amino acids • The assembled structure of the ribosome creates three pockets for the binding of two molecules of tRNA. •The far left pocket is the Exit site or E site •It binds the deacylated tRNA (no amino acid attached) • The one in the middle is referred to as the peptidyl or the P site: • it binds to the tRNA holding the growing chain of polypeptide. • The site on the right is termed the amino acyl, or the A site, •it binds to the incoming tRNA molecule. 16 /35 17 /35 18 Mechanism of Translation •Three steps of translation: – Initiation: sets the stage for polypeptide synthesis. – Elongation: causes the sequential addition of amino acids to the polypeptide chain in a colinear fashion as determined by the sequence of mRNA. – Termination: Brings the polypeptide synthesis to a halt. 20 /35 Initiation !!he initiation codon is an AUG ! is towards the 5’ end of the mRNA molecule that is being translated. ! NOT the first 3 nucleotides! ! It determines the reading frame. !In prokaryotes, there is a conserved region about 7 nucleotides upstream from the initiating AUG: ! this region contains a 6-nucleotide sequence ! Shine-Dalgarno box: AGGAGG. !The Shine-Dalgarno sequence is complementary to a region at the 3’ end of the 16 rRNA of the small subunit; ! base pairing between these complementary sequences stabilizes the binding of the small ribosomal subunit to the mRNA for proper assembly. 21 /35 22 /35 Initiation: continued ! In prokaryotes, the first AUG is recognized by a special tRNA (tRNAfMet) carrying a modified methionine: formyl methionine. ! The large subunit of the ribosome now attaches to the small subunit, to complete the initiation process. ! In eukaryotes, the small ribosomal unit binds first to the methylated cap (7-methyl guanosine) at the 5’ end of the mRNA. ! It then migrates to the initiation site, usually the first AUG it encounters as it scans the mRNA in the 5’ to 3’ direction. ! In eukaryotes, the methionine need not be modified. 23 /35 Fig. 14.12 Initiation of protein synthesis in prokaryotes 24 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Translation Initiation in Eukaryotes 25 Elongation • At the start of elongation, the mRNA is bound to the complete two subunit ribosome, – with the initiating tRNA in the P site, – and the A site free for binding to the next tRNA. • The ribosome moves along the mRNA in a 5’ to 3’ direction, in a step-wise process, recognizing each subsequent codon. • The peptidyl transferase enzyme then catalyzes the formation of a peptide bond between – the free N terminal of the amino acid at the A site, – and the Carboxyl end of the amino acid at the P site, which is actually connected to the tRNA. • This disconnects the tRNA fMet from the amino acid, and the tRNA at the A site now carries two amino acids, – with a free N terminal and the Carboxyl terminal of the second aa connected to its tRNA. 26 /35 Translation Elongation 27 Fig. 6.13 The formation of a peptide bond between the first two amino acids of a polypeptide chain is catalyzed on the ribosome by peptidyl transferase 28 Chain Elongation:Translocation • 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. • 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. 29 Chain Elongation: Translocation • Elongation and translocation are similar in eukaryotes, except for differences in number and type of elongation factors and the exact sequence of events. • 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 attached at a given moment (Figure 14.15). 30 Fig. 6.14 Diagram of a polysome, a number of ribosomes each translating the same mRNA sequentially 31 Termination • When the ribosome encounters a stop codon, – there is no tRNA available to bind to the A site of the ribosome, – instead a release factor binds to it. • The details are not very clear, but once the release factor binds, the ribosome unit falls apart, – releasing the large and small subunits, – the tRNA carrying the polypeptide is also released, freeing up the polypeptide product. 32 /35 Fig. 14.15 Termination of translation Release Factor Release Factor Release Factor 33 35 /35