Vocabulary

1. Vocabulary • Replication -- copying DNA before cell division • Transcription -- making an RNA copy (messenger RNA or mRNA) of DNA. Note -- Transcription involves copying in the same language (e.g., court transcription). • Translation -- making a protein from the mRNA. Note -- The nucleic acid language is being translated into the protein language. DNA ---------> DNA DNA ---------> RNA ----------> Protein RNA ---------> DNA replication transcription translation reverse transcription

2. Clicker question DNA is transcribed into RNA in the _______.RNA is translated into protein in the _______. 1) Endoplasmic reticulum, Golgi apparatus 2) Golgi apparatus, Endoplasmic reticulum 3) Nucleus, cytoplasm 4) Cytoplasm, nucleus 5) Nucleus, nucleus 6) Cytoplasm, cytoplasm

3. mRNA is spliced before leaving the nucleus to remove non-coding regions (introns)

4. Definitions Intron -- Non-coding intervening sequence Exon -- Coding, or expressed, sequencesBacterial genes don’t have introns

5. Alternative splicing gives rise to variant proteins in different cell types • Alternative splicing allows eukaryotes to pack more information into each gene. • Genes for new proteins can evolve rapidly by combining exons.

6. Proteins are often made from independently-folded domains encoded on separate exons Fab: The antigen*-binding fragment of an antibody. Four immunoglobulin domains arranged as two domains per polypeptide chain. *Antigen: protein, small molecule or carbohydrate recognized by an antibody. CD4: The T cell co-receptor and HIV receptor. Four immunoglobulin-like domains arranged in tandem.

7. Only mature, correctly processed, RNA leaves the nucleus RNA must be spliced before leaving the nucleus. This is a problem for HIV, which needs to package incompletely spliced or unspliced RNA. Later we will discuss the mechanism HIV has evolved to get around this problem.

8. HOW DID THIS DIFFERENCE (introns versus no introns) EVOLVE? Evolutionary flexibility A way to control gene expression BUT Cell has to maintain larger genome Cell throws out a large fraction of the RNA it synthesizes

9. Maybe early cells had introns, which were lost by prokaryotes as they evolved to reproduce more rapidly and efficiently. • Simple eukaryotes that reproduce rapidly (e.g., some yeasts) have very few (and short) introns. • Maybe introns were originally parasitic mobile genetic elements that invaded an early eukaryote. Host cells then replicated the “selfish DNA” along with their own DNA, and modern eukaryotes never bothered to get rid of it. WHY INTRONS?

10. We can translate because we know the code

11. The genetic code is triplet, unpunctuated, and non-overlapping Codon: a group of three consecutive nucleotides encoding one amino acid

12. Clicker question How did Sydney Brenner rule out the possibility of an overlapping triplet code? 1) By noticing that an overlapping code would restrict which amino acids were next to each other, but finding no evidence for restrictions in protein sequences. 2) By realizing that an overlapping code would produce too many stop codons. 3) By sequencing RNA and proteins. By looking up the answer in Wikipedia. By asking a Bi1 student.

13. The genetic code is degenerate In addition, there are three stop codons (UAA, UGA and UAG) and one start codon (AUG)*. *AUG codes for methionine, which can also be in the middle of a protein.

14. Clicker question In which of the following would one find exceptions to the genetic code? 1) Mitochondria 2) Some yeast 3) Some ciliates 4) Some bacteria 5) Bi1 students 6) All of the above

15. Most mitochondrial proteins are encoded by nuclear genes. However, mitochondria contain their own DNA, as well as a complete transcription and translation system for producing proteins from these genes. Clicker question Why do mitochondria contain their own DNA? 1) Proteins cannot pass through the double membrane of a mitochondrion, therefore they have to be synthesized inside it. 2) Mitochondria originated from bacteria that were engulfed by an ancestral eukaryotic cell. They survived, gradually losing some, but not all, of their genes. 3) All intracellular organelles contain DNA, so mitochondria share this property with the endoplasmic reticulum and Golgi apparatus. 4) Because mitochondria are continuously dividing and fusing during the lifetime of the cell, localized transcription and translation allows them to respond quickly to metabolic needs of the cell. 5) To confuse Bi1 students.

16. Clicker question Why do mitochondria contain their own DNA? Mitochondria originated from bacteria that were engulfed by an ancestral eukaryotic cell. They survived, living in symbiosis with their host, gradually losing some, but not all, of their genes.

17. Only one of the three reading frames encodes the real message. It is usually obvious which is the correct reading frame (open reading frame or ORF) because the other two reading frames are likely to contain stop codons. mRNA can be read in 3 different reading frames

18. Clicker question How would you design a system to pair an amino acid with its correct codon? 1) Evolve ≥20 different amino acid-carrying proteins that recognize triplet codons. 2) Design amino acids that bind specifically to their codon(s). 3) Evolve enzymes to attach amino acids to triplets of RNA. 4) Evolve ≥20 different “adapter” RNAs, each of which uses one region to recognize a particular codon and another region to bind the amino acid corresponding to that codon.

19. Transfer RNAs (tRNA) serve as adaptors to link amino acids to codons There is more than one tRNA for most amino acids. tRNAs can tolerate a mismatch at the third position of the codon (wobble base).

20. Why do tRNAs fold into a clover leaf structure? Clicker question 1) Three hairpins are formed due to short regions of complementary RNA, which basepair. 2) Some of the bases in tRNA are hydrophobic, so these form a hydrophobic core that is shielded from water. 3) The clover leaf structure maximizes the possible interactions between oppositely-charged basepairs. 4) tRNAs evolved from clover plants.

21. Translation Eukaryotic ribosomes: 2 amino acids added per second Prokaryotic ribosomes: 20 amino acids added per second

22. Transfer RNAs (tRNA) serve as adaptors to link amino acids to codons  = pseudouridine; one of >100 different modified nucleosides in RNA (but not mRNA). Made post-transcriptionally by -synthase.

23. Translation in more detail

24. Incorporating unnatural amino acids into proteins In vivo nonsense suppression Nonsense mutations (those that introduce stop codons) can be “suppressed” by a suppressor tRNA (anti-codon recognizes a stop codon, but tRNA is coupled to an amino acid) Use organic chemistry to couple an unnatural amino acid to a suppressor tRNA Deliver unnatural amino acids to the ribosome Extend the genetic code beyond 20 amino acids! See Dennis Dougherty and David Tirrell (CCE) for more information

25. Unnatural amino acids that have been added to the genetic code of prokaryotes and eukaryotes (Xie & Schultz, Nat Reviews Mol Cell Biol, 2006)

26. The ribosome is a protein manufacturing machine Travels along the mRNA, captures complementary tRNA molecules and holds them in position, then covalently links the amino acids they carry to form a protein chain. Ribosomes contain >80 different proteins and 4 RNAs (ribosomal RNA or rRNA). Amazingly, ribosomes have been crystallized, and we now have atomic resolution structures showing the positions of all (except hydrogen) atoms of these enormous (several million Dalton) machines.

27. RNA, not protein, is responsible for catalysis in a ribosome. The ribosome is a “ribozyme” (an RNA enzyme). L1, a ribosomal protein, is shown for scale. Ribosomes are 2/3 RNA, 1/3 protein

28. Translating polyribosomes

29. Bil Clemons, Assistant Professor of Chemistry, Caltech, solved a ribosome structure in Ramakrishnan’s lab as a graduate student.

31. Some proteins fold as they come off the ribosome

32. Membrane and secreted proteins are co-translationally inserted into the endoplasmic reticulum membrane

33. EXTRA SLIDES & MOVIES

34. tRNA synthetase couples an amino acid to its tRNA, then the tRNA anticodon base pairs with the appropriate codon on the mRNA

35. View of a cell -- look at all the ribosomes! ER: yellow membrane-bound ribosomes: blue free ribosomes: orange microtubules: bright green mitochondria: dark green dense core vesicles: blue clathrin-negative vesicles: white clathrin-positive compartments and vesicles: red clathrin-negative compartments and vesicles: purple 500 nm Marsh et al. (2001) PNAS 98: 2399-2406

36. RNA transcripts are handled differently in eukaryotes and prokaryotes

37. RNA splicing mechanism movie You don’t need to know all of the details the splicing mechanism. You do need to know the definitions of “intron” and “exon” and understand that signals on the RNA attract a splicing machine (spliceosome), which precisely removes the intron.

38. Initiator tRNA searches for the start signal along mRNA

39. Clicker question Propose a mechanism by which the highlighted areas could cause transcription to stop. 5) The first two sequences are inverted repeats of each other, thus a hairpin, which is a signal for termination.

40. How can tRNAs and other hairpin-containing RNAs be transcribed? Need stretch of Uridines in RNA after hairpin -- otherwise RNA polymerase slows down rather than terminates. Termination not precise -- transcripts contain different lengths of 3’ untranslated regions. Sometimes terminator or anti-terminator proteins are involved.