PROTEIN SYNTHESIS, STRUCTURE AND FUNCTION YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE

1. PROTEIN SYNTHESIS, STRUCTURE AND FUNCTION YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE THE DEPARTMENT OF MEDICAL BIOLOGY ASST. PROF. DR. ENDER ŞİMŞEK

3. Protein Synthesis • The production or synthesis of proteinsis composed of two phases:Transcription & Translation. • mRNA must be processed (before it leaves the nucleus of eukaryotic cells).

4. Protein synthesis: *Aminoacids *tRNAs *mRNA *AminoacyltRNAsynthetases *Ribosomes *Initiation, elongationandreleasingfactors *ATP’sandGTP’s

5. PROTEIN SYNTHESIS: 1-Activation of aminoacids: ATP, t-RNA Aminoacid Aminoacylt-RNA senthetase 2-Initiation of protein synthesis: m-RNA (start codon, AUG) Ribosome, GTP Initiating t-RNA (AUG anticodon) InitiationFactors (IF1, IF2, IF3)

6. PROTEIN SYNTHESIS: 3-Elongation of protein synthesis: ElongationFactors(EF1, EF2) GTP 4-Termination of protein synthesis: m-RNA stop codon (UAA, UAG, UGA) ReleasingFactors(RF1, RF2, RF3)

7. RIBOSOMES In addition to the APE sites,there is an mRNA binding groove that holds onto the message being translated.

8. Amino-acyl tRNAsynthetases: ***One synthetase for each amino acid ***A single synthetase may recognize multiple tRNAs for the same amino acid

9. Amino-acyl tRNAsynthetases: ***One synthetase for each amino acid ***A single synthetase may recognize multiple tRNAs for the same amino acid Two classes of synthetase: Different 3-dimensional structures, Differ in which side of the tRNA they recognize and how they bind ATP. Class I - monomeric, acylates the 2’OH on the terminal ribose Arg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val Class II - dimeric, acylatesthe 3’OH on the terminal ribose Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr

10. Class I - monomeric, acylates the 2’OH on the terminal ribose Arg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val Class II - dimeric, acylates the 3’OH on the terminal ribose Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr

12. Initiation of Translation • In prokaryotes, ribosomes bind to specific translation initiation sites. • (Shine Dalgarno Sequence: 5‘-UAAGGAGG-3‘) • There can be several different initiation sites on a messenger RNA: a prokaryotic mRNA can code for several different proteins.

13. Shine-DalgarnoSequence

14. Initiation of Translation • In prokaryotes, ribosomes bind to specific translation initiation sites. • (Shine Dalgarno Sequence: 5‘-UAAGGAGG-3‘) • There can be several different initiation sites on a messenger RNA: a prokaryotic mRNA can code for several different proteins. • Translation begins at an AUG codon. • The modified amino acid N-formyl methionine is always the first amino acid of the new polypeptidein prokaryotes.

15. Initiation of Translation • In eukaryotes, ribosomes bind to the 5’ cap, then move down the mRNA until they reach the first AUG, the codon for methionine. Translationstarts from this point. • (MarlynKozak Sequence: 5’-ACCAUGG-3’)

16. Initiation of Translation • In eukaryotes, ribosomes bind to the 5’ cap, then move down the mRNA until they reach the first AUG, the codon for methionine. Translationstarts from this point. • (MarlynKozak Sequence: 5’-ACCAUGG-3’) • Eukaryotic mRNAs code for only a single gene. • Translation does not start at the first base of the mRNA. There is an untranslated region at the beginning of the mRNA, the 5’untranslated region (5’ UTR).

17. Initiation… • The initiation process involves first joining the mRNA, the initiator methionine-tRNA, and the small ribosomal subunit.

18. Initiation… • The initiation process involves first joining the mRNA, the initiator methionine-tRNA, and the small ribosomal subunit. • Several “initiation factors”--additional proteins--are also involved. The large ribosomal subunit then joins the complex.

19. Elongation • The ribosome has 2 sites for tRNAs, called P and A. • The initial tRNA with attached amino acid is in the P site.

20. Elongation • A new tRNA, corresponding to the next codon on the mRNA, binds to the A site. • The ribosome catalyzes a transfer of the amino acid from the P site onto the amino acid at the A site, forming a new peptide bond.

21. Elongation • The ribosome then moves down one codon. • The empty tRNA at the P site is displaced off the ribosome, • and the tRNA that has the growing peptide chain on it is moved from the A site to the P site.

22. Elongation • The process is then repeated: • the tRNA at the P site holds the peptide chain, and a new tRNA binds to the A site. • the peptide chain is transferred onto the amino acid attached to the A site tRNA. • the ribosome moves down one codon, displacing the empty P site tRNA and moving the tRNA with the peptide chain from the A site to the P site.

23. Elongation

24. Termination • Three codons are called “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon.

25. Termination • Three codons are called “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon. • When the ribosome reaches a stop codon, there is no tRNA that binds to it.

26. Termination • Three codons are called “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon. • When the ribosome reaches a stop codon, there is no tRNA that binds to it. • Instead, proteins called “release factors” bind, and cause the ribosome, the mRNA, and the new polypeptide to separate.

27. Termination • Note that the mRNA continues on past the stop codon. The remaining portion is not translated: it is the 3’untranslated region (3’ UTR).

28. Translation: Initiation • mRNA binds to a ribosome, and the transfer RNA corresponding to the START codon binds to this complex. Ribosomes are composed of 2 subunits (large and small), which come together when the messenger RNA attaches during the initiation process.

29. Translation: Elongation • Elongation: the ribosome moves down the messenger RNA, adding new amino acids to the growing polypeptide chain. • The ribosome has 2 sites for binding transfer RNA. The first RNA with its attached amino acid binds to the first site, and then the transfer RNA corresponding to the second codon bind to the second site.

30. Translation: Elongation • The ribosome then removes the amino acid from the first transfer RNA and attaches it to the second amino acid. • At this point, the first transfer RNA is empty: no attached amino acid, and the second transfer RNA has a chain of 2 amino acids attached to it.

31. Translation: Termination • The elongation cycle repeats as the ribosome moves down the messenger RNA, translating it one codon and one amino acid at a time. • The process repeats until a STOP codon is reached.

37. mRNA A U G C U A C U U C G Ribosomes Large subunit P Site A Site Small subunit

38. aa2 aa1 2-tRNA 1-tRNA G A U U A C Initiation anticodon A U G C U A C U U C G A hydrogen bonds codon mRNA

39. aa3 3-tRNA G A A Elongation peptide bond aa1 aa2 1-tRNA 2-tRNA anticodon U A C G A U A U G C U A C U U C G A hydrogen bonds codon mRNA

40. aa3 3-tRNA G A A aa1 peptide bond aa2 1-tRNA U A C (leaves) 2-tRNA G A U A U G C U A C U U C G A mRNA Ribosomes move over one codon

41. aa4 4-tRNA G C U peptide bonds aa1 aa2 aa3 2-tRNA 3-tRNA G A U G A A A U G C U A C U U C G A A C U mRNA

42. aa4 4-tRNA G C U peptide bonds aa1 aa2 aa3 2-tRNA G A U (leaves) 3-tRNA G A A A U G C U A C U U C G A A C U mRNA Ribosomes move over one codon

43. aa5 5-tRNA U G A peptide bonds aa1 aa2 aa4 aa3 3-tRNA 4-tRNA G A A G C U G C U A C U U C G A A C U mRNA

44. aa5 5-tRNA U G A peptide bonds aa1 aa2 aa3 aa4 3-tRNA G A A 4-tRNA G C U G C U A C U U C G A A C U mRNA Ribosomes move over one codon

45. aa5 aa4 Termination aa199 aa200 aa3 primary structure of a protein aa2 aa1 terminator or stop codon 200-tRNA A C U C A U G U U U A G mRNA

46. aa5 aa4 aa3 aa2 aa199 aa1 aa200 End Product –The Protein! • The end products of protein synthesis is a primary structure of a protein • A sequence of amino acid bonded together by peptide bonds

47. DNA TRANSCRIPTION 1 3 4 2 5 RNA is transcribed from a DNA template. 3 Poly-A RNA transcript RNA polymerase 5 Exon RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Cap NUCLEUS Amino acid FORMATION OF INITIATION COMPLEX AMINO ACID ACTIVATION tRNA CYTOPLASM After leaving the nucleus, mRNA attaches to the ribosome. Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Growing polypeptide mRNA Activated amino acid Poly-A Poly-A Ribosomal subunits Cap 5 TRANSLATION C C A U A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) A E A C Anticodon A A A U G U G G U U U A Codon Ribosome A summary of transcription and translation in a eukaryotic cell Figure 17.26

48. PROTEİNS • Structure: • Primer • Seconder • Tertiary • Quaterner • Bonds: • Peptidbonds • Disülfidbonds • Hidrojen bonds • İonicbonds • Apolarbonds

49. Protein Functions • Protein Binding • Protein conformation • Antibody • Enzyme and substrate • Catalytic Reaction • Kinase and phosphatase • GTPase, GEF, GAP • Motor protein • Membrane-bound protein

50. Amino AcidsProteins are made of 20 amino acids linked by peptide bonds Hydrophilic Hydrophobic