Protein Synthesis

1. Protein Synthesis The Building of Proteins from a Nucleic Acid Template

2. DNA • Provides the Template. • Information contained in the SEQUENCE of N-bases found along the DNA molecule. • Transferring that information into an Amino Acid SEQUENCE is the trick. A G C C T A G G G A T A G T C G G A T C C C T A T C

3. A 2-step Process • Transcription DNA RNA • Translation RNA Protein

4. Transcription • Production of RNA. • RNA = Ribonucleic Acid. • Ribose Sugar. • Single Stranded Molecule. • Uracil instead of Thymine.

5. Transcription • RNA Polymerase • Attaches at a Promoter – a sequence of the DNA that indicates where a gene starts. • With the help of Transcription Factors.

6. Transcription • A “window” in the DNA is opened – ultimately, a gene. • RNA Nucleotides are added (from 5’ to 3’) in accordance with the DNA parent template (which is read from 3’ to 5’).

7. Transcription • Complimentary Base Pairing. • DNA RNA G - C C - G T - A A - U

8. Transcription • Elongation of a single stranded RNA molecule. • The DNA window closes behind.

9. Transcription • Termination – a sequence of bases is reached signaling RNA Polymerase to release. • The “gene” closes • The RNA transcript is released.

10. RNA Processing In Eukaryotic cells, things get a little more complex: • Several types of RNA polymerase are present – each with a slightly different function – I, II, and III. • A larger mRNA is transcribed…

11. A A U A A A RNA Processing • During Transcription, RNA polymerase II transcribes MORE than just the protein-encoding part of the gene. • Untranslated Regions (UTRs) are transcribed at the 5’ and 3’ ends. • The 3’ UTR is called a Polyadenylation signal. 3’ UTR 5’ UTR

12. 5’cap Poly-A tail A A U A A A RNA Processing • AFTER Transcription is terminated, additional alterations are made to the ends of this “pre-mRNA” strand. • A 5’ cap at the leading (5’) end (composed of a Guanine-like nucleotide). • A poly-A tail at the trailing (3’) end (composed of many Adenine nucleotides).

13. 5’ Cap and Poly-A Tail These END pieces serve several functions: 1. Help “ferry” the eventual mRNA transcript out of the nucleus. 2. Protect the protein-encoding area from degradation. 3. Help attach the proper end (5’) of the transcript to the place where Translation will take place – the Ribosome.

14. 5’cap Poly-A tail A A U A A A Further RNA Processing • RNA splicing – removes portions of the transcript. • Removed segments are called INTRONS. • The remaining coding segments are called EXONS. Introns Exons

15. Further RNA Processing • INTRONS are recognized by snRNPs… • “small nuclear RiboNucleoProteins”. • snRNPs cut out INTRONS and join the adjacent EXONS. • This occurs in the nucleus.

16. 5’cap 5’cap Poly-A tail Poly-A tail A A U A A A A A U A A A Further RNA Processing • The resulting molecule is the RNA transcript proper (no longer the pre-mRNA).

17. Why? • Introns may be vestigial. • Introns may have regulatory roles. • Alternative RNA splicing – more than one polypeptide from a single gene. • More chances of unique Exon combinations (taking into account “crossing over” during meiosis). • (Cross-overs at introns won’t disrupt exons).

18. 3 Types of RNA • mRNA = Messenger RNA • The “blueprint” • rRNA = Ribosomal RNA • The “workbench” • tRNA = Transfer RNA • The “truck”

19. mRNA – messenger RNA • The “message” – the blueprint for the production of a polypeptide – a protein. • But there’s only 4 N-bases, and 20 amino acids! • What’s the code?

20. The Genetic Code • Triplet Base Code. • Every 3-letter word in the RNA Transcript is a CODON.

21. The Genetic Code • Each CODON codes for a single Amino Acid.

22. The Genetic Code

23. rRNA – Ribosomal RNA • The location for protein synthesis – the workbench on which a polypeptide is built. • rRNA makes up a RIBOSOME. • Ribosomes have 2 sub-units. Large subunit Small subunit

24. rRNA – Ribosomal RNA • The Large subunit has several binding sites:

25. tRNA – Transfer RNA • The “transfer-er” – the truck that brings individual Amino Acids to the workbench for the production of a polypeptide – a protein. • With a driver – the Anticodon.

26. tRNA – Transfer RNA • Each tRNA carries a specific Amino Acid to the Ribosome. • It “knows” when to drop this Amino Acid off by… • COMPLIMENTARY BASE PAIRING of its Anticodon with the corresponding Codon found on the mRNA.

27. tRNA – Transfer RNA • Aminoacyl-tRNA synthetase = the enzyme responsible for hooking Amino Acids to tRNAs. • Aminoacyl tRNA = truck with cargo.

28. Translation • Translating the RNA sequence data into Protein sequence data. • N-bases Amino Acids • In the cytoplasm of Eukaryotic cells.

29. Translation • Initiation - All 3 types of RNA come together. • This requires some energy (provided by Guanosine Triphosphate -- GTP).

30. Translation • Elongation – Amino Acids are added with the help of elongation factors (proteins). • Codon meets Anticodon • at the ‘A’ binding site. 2. A Peptide Bond Forms between adjacent Amino Acids

31. Translation • Then, a shift in the Ribosome (a “Translocation”) moves the empty tRNA to the exit (E-site), opening the A-site for the next tRNA.

32. Translation • Termination… • Continued elongation until a “STOP” codon is reached (UAG, UAA, or UGA). • A release factor (protein) binds to this codon, effectively releasing the polypeptide. • The mRNA, tRNA, and rRNA separate.

33. Protein Synthesis Primary Structure Of the Protein

34. Protein Synthesis • In response to a need for a particular protein – need it NOW. • The same gene can be transcribed by several RNA polymerase molecules simultaneously…allowing faster protein production. • Polyribosomes – strings of Ribosomes translate a single mRNA simultaneously – allowing faster protein production.

35. Polyribosomes

36. Bound and Free Ribosomes • Free-floating Ribosomes are the sites for free-floating protein production. • Ribosomes bound to membranes are the sites for membrane-bound and secretory proteins.

37. Review – Fig. 17.26

38. Point Mutations • Point Mutations occur at single nucleotides along DNA’s template strand. • Substitutions – the replacement of a pair of nucleotides at a particular location. • Silent Mutations. • Not-So-Silent Mutations. • Missense • Nonsense

39. Substitution Mutations • Original Sequence: • Silent Mutation: • Missense Mutation: • Nonsense Mutation:

40. Point Mutations • Insertions & Deletions – adding or removing bases along the sequence. • Results in a FRAME SHIFT because all other codons “downstream” are affected. • Can reverse if there are 3 insertions or 3 deletions.

41. Insertions & Deletions • Original Sequence: • Insertion/nonsense: • Deletion/missense: • 3-Nucleotide Insertion/Deletion (missense):

42. Assignment: • Review chapters 16 & 17! Transcription: http://www.ncc.gmu.edu/dna/mRNAanim.htm http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html Translation: http://www.ncc.gmu.edu/dna/ANIMPROT.htm http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a3.html Overall: http://www.johnkyrk.com/DNAtranslation.html