MB206

1. MB206 Microbial Biotechnology

2. Important Ms Angelia Teo @ Ms Teo Swee Sen http://mb206.wikispaces.com/MB angeliaucsi@gmail.com

3. Wikispaces • Tools • Discussion • Update new information

4. 1st step – open wikispaces

5. Using your ID as Username

13. What you need to do after this … …

14. Important • Quiz 10% (3 June 2009) • Mid-term 20% (1 July 2009) • Assignments/Tutorials 13% + 2% • Lab 15% • Final Exam 40% • Additional 5%

15. NUCLEIC ACID

17. RNA similar to DNA both nucleic acids but - • RNA is single stranded • RNA has ribose sugar • RNA has uracil in place of thymine

18. A Comparison of DNA and RNA

19. DNA Replication in Bacteria • The DNA replicatessemiconservatively: - Each strand in dsDNA serves as a template for synthesis of a new complementary strand. - Result: daughter dsDNA molecule - contains one old polynucleotide strand and one newly synthesized strand. • Replication of chromosomal DNA in bacteria starts at a specific chromosomal site called theorigin of replicationand proceedsbi-directionallyuntil the process is completed. X Y .

20. Messenger RNA (mRNA) Ribosome: contains ribosomal RNA (rRNA) catalytic site large subunit 1 2 tRNA/amino acid binding sites small subunit Transfer RNA (tRNA) attached amino acid anticodon

21. by Angelia Teo (Jan 09)

22. Central Dogma of Molecular Biology How does the sequence of a strand of DNA correspond to the amino acid sequence of a protein? • DNA codes for RNA production. • RNA codes for protein production. • Protein does not code protein, RNA • or DNA production. • The end. • Or in the words of Francis Crick: Once information has passed into • protein, it cannot get out again!

23. Revision of the "Central Dogma" • CAN go back from RNA to DNA (reverse transcriptase) • RNA can also make copies of itself (RNA polymerase) • Still NOT possible from Proteins back to RNA or DNA • Not known mechanisms for proteins making copies of themselves.

24. by Angelia Teo (Jan 09)

25. Transcription & Translation Prokaryotic vs Eukaryotic cells In a prokaryotic cell, which does not contain a nucleus, this process happens at the same time. In Eukaryotic cells, occur at different cell compartments. Prokaryotic cell Eukaryotic cell

26. Transcription • The DNA-directed synthesis of RNA is called transcription. • Transcription produces RNA molecules that are complimentary copies of one strand of DNA. • Only one of the dsDNA strands can serve as template for synthesis of a specific mRNA molecule. • mRNAs transmit information from DNA, and each mRNA in bacteria function as a template for synthesis of one or more specific proteins.

27. (a) Initiation DNA gene 1 gene 2 gene 3 RNA polymerase DNA promoter RNA polymerase binds to the promoter region of DNA near the beginning of a gene, separating the double helix near the promoter.

28. (b) Elongation RNA DNA template strand RNA polymerase travels along the DNA template strand (blue), catalyzing the addition of ribose nucleotides into an RNA molecule (pink). The nucleotides in the RNA are complementary to the template strand of the DNA.

29. (c) Termination termination signal At the end of a gene, RNA polymerase encounters a DNA sequence called a termination signal. RNA polymerase detaches from the DNA and releases the RNA molecule.

30. Conclusion of transcription RNA After termination, the DNA completely rewinds into a double helix. The RNA molecule is free to move from the nucleus to the cytoplasm for translation, and RNA polymerase may move to another gene and begin transcription once again.

31. Translation • The process by which the nucleotide sequence of an mRNA molecule determines the primary amino acid sequence of a protein. • Ribosomesare complexes of ribosomal RNAs (rRNAs) and several ribosomal proteins. • Ribosomes with the aid oftransfer RNAs (tRNAs),amino-acyl tRNA synthesases, initiation factors and elongation factors are all involved in translation of each mRNA into corresponding polypeptide (protein).

32. Translation • Initiated at an AUG codon for methionine. • Codons are translated sequentially in mRNA from 5' to 3'. • The corresponding polypeptide chain / protein is assembled from the amino terminus to carboxy terminus. • The sequence of amino acids in the polypeptide is, therefore, co-linear with the sequence of nucleotides in the mRNA and the corresponding gene.

33. Initiation: second tRNA binding site amino acid catalytic site tRNA methionine tRNA large ribosomal subunit first tRNA binding site initiation complex mRNA small ribosomal subunit The initiation complex binds to an mRNA molecule. The methionine (met) tRNA anticodon (UAC) base-pairs with the start codon (AUG) of the mRNA. A tRNA with an attached methionine amino acid binds to a small ribosomal subunit, forming an initiation complex The large ribosomal subunit binds to the small subunit. The methionine tRNA binds to the first tRNA site on the large subunit. Elongation: catalytic site catalytic site initiator tRNA detaches peptide bond ribosome moves one codon to right The second codon of mRNA (GUU) base-pairs with the anticodon (CAA) of a second tRNA carrying the amino acid valine (val). This tRNA binds to the second tRNA site on the large subunit. The catalytic site on the large subunit catalyzes the formation of a peptide bond linking the amino acids methionine and valine. The two amino acids are now attached to the tRNA in the second binding position. The "empty" tRNA is released and the ribosome moves down the mRNA, one codon to the right. The tRNA that is attached to the two amino acids is now in the first tRNA binding site and the second tRNA binding site is empty. Termination: catalytic site completed peptide stop codon This process repeats until a stop codon is reached; the mRNA and the completed peptide are released from the ribosome, and the subunits separate. The catalytic site forms a new peptide bond between valine and histidine. A three-amino-acid chain is now attached to the tRNA in the second binding site. The tRNA in the first site leaves, and the ribosome moves one codon over on the mRNA. The third codon of mRNA (CAU) base-pairs with the anticodon (GUA) of a tRNA carrying the amino acid histidine (his). This tRNA enters the second tRNA binding site on the large subunit.

34. Initiation: amino acid methionine tRNA initiation complex small ribosomal subunit A tRNA with an attached methionine amino acid binds to a small ribosomal subunit, forming an initiation complex.

35. tRNA mRNA The initiation complex binds to an mRNA molecule. The methionine (met) tRNA anticodon (UAC) base-pairs with the start codon (AUG) of the mRNA.

36. second tRNA binding site catalytic site large ribosomal subunit first tRNA binding site The large ribosomal subunit binds to the small subunit. The methionine tRNA binds to the first tRNA site on the large subunit.

37. Elongation: catalytic site The second codon of mRNA (GUU) base-pairs with the anticodon (CAA) of a second tRNA carrying the amino acid valine (val). This tRNA binds to the second tRNA site on the large subunit.

38. peptide bond The catalytic site on the large subunit catalyzes the formation of a peptide bond linking the amino acids methionine and valine. The two amino acids are now attached to the tRNA in the second binding position.

39. catalytic site initiator tRNA detaches ribosome moves one codon to right The "empty" tRNA is released and the ribosome moves down the mRNA, one codon to the right. The tRNA that is attached to the two amino acids is now in the first tRNA binding site and the second tRNA binding site is empty.

40. catalytic site The third codon of mRNA (CAU) base-pairs with the anticodon (GUA) of a tRNA carrying the amino acid histidine (his). This tRNA enters the second tRNA binding site on the large subunit.

41. The catalytic site forms a new peptide bond between valine and histidine. A three-amino-acid chain is now attached to the tRNA in the second binding site. The tRNA in the first site leaves, and the ribosome moves one codon over on the mRNA.

42. Termination: completed peptide stop codon This process repeats until a stop codon is reached; the mRNA and the completed peptide are released from the ribosome, and the subunits separate.

43. The Genetic code The "universal" genetic code employed by most organisms is a triplet code and it determines how the nucleotides in mRNA specify the amino acids in the polypeptide. • 61 of 64 possible trinucleotides (codons) encode specific amino acids. • 3 remaining codons (UAG, UAA or UGA) code for termination of translation (nonsense codons = do not specify any amino acids) • Exceptions: • UGA as a tryptophan codon in some species of Mycoplasma and in mitochondrial DNA. • Few codon differences in mitochondrial DNAs from yeasts, Drosophila, and mammals.

44. Gene expression occurs in 2 steps: Transcriptionof the information encoded in DNA into a molecule of RNA Translation of the information encoded in mRNA into a defined sequence of amino acids in a protein.