MOLECULAR GENETICS TRANSCRIPTION & TRANSLATION

1. MOLECULAR GENETICSTRANSCRIPTION & TRANSLATION

2. ANNOUNCEMENTS • EXAM 2 is MONDAY (JULY 10th) • Same format as Exam 1 • Bring #2 pencil(s) and eraser • Bring STUDENT ID • No calculators, cells phones or hats • ExtendedOffice hours: • FRIDAY 4 – 6 pm • Practice Exams (II) posted on web site!

3. ANNOUNCEMENTS • Use your student CD to help visualize transcription/translation!

4. OBJECTIVES • Know processes that make up transcription • Recognize role of RNA Polymerase • Understand significance of promoter and terminator regions of DNA • Explain how transcribed RNA is modified prior to exiting nucleus

5. OBJECTIVES • Understand process of translation • Recognize role of mRNA, rRNA and tRNA • Understand how proteins may be modified prior to use

6. THE CENTRAL DOGMA Transcription: • Synthesis of RNA under direction of DNA • Occurs in nucleus (eukaryotes) • Players include: • DNA • Messenger RNA (mRNA) • RNA Polymerase

7. THE CENTRAL DOGMA Translation: • Synthesis of polypeptides under direction of mRNA • Occurs in cytoplasm • Ribosomes • Major players include: • mRNA • Transfer RNA (tRNA) • Ribosomes (rRNA)

8. EUKARYOTIC TRANSCRIPTION Consists of Three Stages: • Initiation: • Attachment of RNA Polymerase to “promoter” region of DNA • Elongation: • Building mRNA complementary to DNA • Termination: • Release of RNA Polymerase & mRNA

9. EUKARYOTIC TRANSCRIPTION • Initiation: • Promoter region • Sequence of A & T on DNA • TATA BOX • Transcription Factors: • Proteins assist binding of RNA Polymerase to promoter • Transcription Initiation Complex • Forms when Transcription Factors & RNA Polymerase bind to promoter region

10. 2. Elongation: Two strands of DNA unwind RNA Polymerase builds mRNA complimentary to DNA (60 bases/sec) Uses NTPs as monomers Ribonucleoside triphosphate DNA double helix reforms after RNA Polymerase passes EUKARYOTIC TRANSCRIPTION base 5’ CH2 1’ 4’ 3’ 2’ 5’ mRNA 3’ RNA 5’ 3’ A U A C G T C 3’ 5’ DNA

11. EUKARYOTIC TRANSCRIPTION

12. EUKARYOTIC TRANSCRIPTION 2. Elongation: • Template Strand: • Only one strand involved in transcription • Codon: • Complimentary nucleotide triplets of mRNA • U substituted for T as compliment for A • 61 codons specify 20 amino acids • 3 codons act as punctuation ONLY

13. EUKARYOTIC TRANSCRIPTION 3. Termination: • When RNA Polymerase transcribes terminator region of DNA, Polymerase releases mRNA • Termination sequence on mRNA is AAUAAA

14. POST TRANSCRIPTIONAL EVENTSModification of mRNA • Transcribed mRNA is modified before leaving nucleus: • Addition of 5’cap • Prevents “unraveling” • Addition of Poly A tail • Prevents “unraveling” • Helps ribosome attach • Assists in export of mRNA from nucleus

15. POST TRANSCRIPTIONAL EVENTSModification of mRNA • Exons • Segments of pre-mRNA that contain information that will be reflected in polypeptide • Introns • Segments of pre-mRNA that separate (intervene) exons

16. POST TRANSCRIPTIONAL EVENTSModification of mRNA Intron 1 Intron 2 DNA Promoter Exon 1 Exon 2 Exon 3 Primary RNA Processed transcript Noncoding regions must be edited out of RNA transcripts.

17. POST TRANSCRIPTIONAL EVENTSModification of mRNA • snRNPS: • Small nuclear ribonucleicproteins • Recognize intron ends and, together with proteins, form a structure called a spliceosome • Spliceosomes remove introns & connect exons together • Ribozymes may also catylyze this process in some organisms

19. Visual Evidence of Transcription RNA Polymerase DNA

20. GENETICS PROBLEMS • SET #2

21. TRANSLATION • Flow of genetic info from mRNA to protein: • Ribosomes • Cytoplasm • Change in cellular language from nucleotide (mRNA) to protein (sequence of amino acids (aa)) • tRNA acts as interpreter

22. PRE TRANSLATIONAL EVENTS Role of Transfer RNA (tRNA): • Before translation begins, tRNA must bind to a specific aa • tRNA: • The interpreter between nucleic acid & protein • Composed of RNA • Has 3-D structure • Includes 3 loops within the molecule • 3’ end attaches to specific aa • Anticodon (2nd loop) compliments the codon on mRNA

24. Early model of tRNA function Amino acid Ser 3’ A C Binding site for amino acid C 5’ Binding site for mRNA codon Serine anticodon A U G 5’ 3’ U C A mRNA Serine codon

25. Revised model incorporating tertiary structure of tRNA Ser 5’ C C A 3’ Amino acid attached to 3’ end Hydrogen bonds Anticodon mRNA A G U 5’ 3’ U C A Codon

26. PRE TRANSLATIONAL EVENTS Role of Transfer RNA (tRNA): • Binding of tRNA & a.a. requires: • An Enzyme • Aminoacyl-tRNA synthetase • ATP

27. TRANSLATION Occurs in Three Steps: • Initiation • Binding of first tRNA, small & large ribosomal subunits to mRNA • Elongation • Lengthening of amino acid chain • Termination • End of synthesis and peptide release

28. TRANSLATION • INITIATION: • Formation of Initiation Complex: • mRNA • Ribosomal Small Subunit • Attaches to mRNA and moves to AUG codon • tRNA • Carrying Methionine • Attaches to the AUG start codon • Protein initiation factors required to assemble the complex

29. TRANSLATION • INITIATION: • Ribosomal Large Subunit: • Attaches and forms “P” site • Holds tRNA with growing peptide chain • An “A” site • Is formed around next codon • The “E” site • Releases tRNA (exit site)

30. TRANSLATION 2.Elongation: • Codon at “A” site forms bond with anticodon of tRNA • Requires energy • Peptide bond forms between polypeptide (on tRNA in “P” site) and aa (in “A” site) • This transfers polypeptide to “A” site • Bond formation catalyzed by a ribozyme

31. TRANSLATION 2.Elongation: • H-bonded mRNA-tRNA unit moves from “A” to “P” site • Requires energy • tRNA occupying “P” site moves to “E” site, where it’s released from ribosome • Elongation continues until stop codon is read by ribosome

32. TRANSLATION 3. TERMINATION: • When stop codon is encountered on mRNA: • Polypeptide is released from tRNA • Ribosomal subunits disengage from each other

33. Micrograph of translation in action Interpretation of micrograph Proteins mRNA Ribosomes RNA polymerase End of gene DNA Start of gene

34. POST TRANSLATIONAL EVENTS • Many synthesized proteins undergo modification before use! • Post-translational events effect protein: • Structure • Activity • Destination

35. POST TRANSLATIONAL EVENTS Protein Structure: • Attachment of sugar, lipids, functional groups • Polypeptide chain may be divided up into smaller units • Protein may require several polypeptide chains • Proteins may require assistance to fold into 3D structure • Assisted by molecular “chaperones”

36. POST TRANSLATIONAL EVENTS Protein Activity: • Proteins may be activated or inactivated through: • Removal of inhibitory peptides • Phosphorylation • Other chemical modifications

37. POST TRANSLATIONAL EVENTS Protein Destination: • Proteins must be sent to proper location • In or outside cell • Proteins destined for locations other than cytoplasm are tagged with signal sequences

38. To cytoplasm To nucleus Newly synthesized proteins Signal sequences mRNA Endoplasmic reticulum Ribosomes To cell membrane (for insertion or secretion) Golgi complex To mitochondrion, chloroplast, or other organelle