1. Bio 178 Lecture 29 DNA and Gene Expression

2. Reading Chapters 14 & 15 Quiz Material Questions on P 300 & 318 Chapters 14 & 15 Quizzes on Text Website (www.mhhe.com/raven7)

3. Outline DNA  DNA Replication Gene Expression  Central Dogma  Major Players  Genetic Code  Transcription

4. Leading and Lagging Strands

5. The Replication Process (Cntd.) Some of the “Other” Enzymatic Activities 2. DNA Primase Synthesizes primers (RNA). 4. DNA Ligase Ligates Okazaki fragments (phosphodiester bond). 3. DNA pol I Removes the primers (*5 ´ to 3´ exonuclease) and replaces them with DNA. 1. DNA Helicase Opens the DNA in front of polymerase. DNA Pol III is the main polymerase.

6. Why Can’t DNA Pol I Remove Primers as a 3 to 5 Exonuclease?

7. E. Coli DNA Replication Enzymes

8. The DNA Replication Fork

9. DNA Replication McGraw-Hill Video

10. DNA Replication in Eukaryotes Similar to E. coli, but involves multiple origins of replication for each chromosome. One Gene/One Polypeptide One gene encodes one polypeptide. Proteins are Composed of Distinct Amino Acid Sequences Sanger, 1953 Sequenced insulin &  showed that proteins are composed of specific amino acid sequences that are always the same for that protein. This, and other work, led to the realization that the gene (specific sequence of nucleotides) determines the amino acid sequence of a protein.

12. Gene Expression - The Central Dogma DNA  RNA  Protein (The Expression of Genes) Stages of Protein Synthesis 1. Transcription DNA  mRNA Occurs in the nucleus. 2. Translation mRNA  Polypeptide Chain Occurs in the cytoplasm.

13. Gene Expression - The Central Dogma

14. Gene Expression - The Major Players Ribosomes Composition 1. 2 subunits - a small and a large: Eukaryotes - 40S and 60S. Prokaryotes - 30S and *50S. 2. Small subunit (Prokaryotes): > 20 proteins and 1 rRNA strand. 3. Large Subunit: > 30 proteins and 2 rRNA strands.

15. Structure of Large Ribosomal Subunit (Prokaryotes) Gray = RNAGold = Protein

16. Ribosomes (Cntd.) Ribosomes are Ribozymes Active site is composed of RNA. The proteins act as a scaffold. Important Sites E, P, and A - where tRNAs bind.

17. Gene Expression - The Major Players (Cntd.) Types of RNA 1. Ribosomal RNA (rRNA) 2. Messenger RNA (mRNA) Site of ribosome where polypeptide is assembled. The single stranded RNA copy of the DNA that carries information from the nucleus to the cytoplasm. 3. Transfer RNA (tRNA) Adapter molecules that pick up specific amino acids and pair them with the complementary sequence of nucleotides on the mRNA.

18. Transfer RNA Structure

19. The Genetic Code How Does the Order of DNA Nucleotides Specify the Order of Amino Acids in Polypeptide? Crick et al., 1961 Hypothesis Genetic code consists of blocks of information (codons). Each codon probably consists of 3 nucleotides that code for 1 amino acid. Reasoning 4 bases - If only 2 bases compose a codon, there are 4 2 (16) possibilities. If 3 bases compose a codon there are 4 3 (64) possibilities. There are 20 aa  3 bases/codon would be enough.

20. Genetic Code - Crick et al. (Cntd.) Question Is the genetic code continuous? Experiment Deleted 1, 2, or 3 nucleotides at a time from viral DNA and tested the effect on transcription. Results Nonsense message was obtained when 1 or 2 nucleotides were deleted (reading frame shifted). 3 deletions - reading frame restored & downstream sequences transcribed correctly. Conclusions This is a triplet code with no “puctuation”.

21. The Genetic Code (Cntd.) Breaking the Code Nirenberg (1961) PolyU mRNA translated in vitro  Polyphenylalanine  UUU codes for phenylalanine. Nirenberg and Leder (1964) Developed an assay (involving radioactive amino acids)  identified what aa 47 of the 64 triplets code for. H. Gobind Khorana Decoded the remaining 17 triplets.

22. The Genetic Code

23. The Genetic Code (Cntd.) Universality Almost all organisms use the same genetic code. Strong evidence for a shared common evolutionary lineage. Important for genetic engineering. Exceptions Mitochondria & chloroplasts - Suggests the change occurred after they became endosymbiotic. Certain protists.

25. Transcription Prokaryotes 1. RNA Polymerase A large enzyme that: (a) Binds to the promoter. (b) Moves down the DNA, untwisting it and breaking the hydrogen bonds between the bases. (c) Pairs complementary nucleotides to the template strand (antisense (-)). Synthesis occurs 5 to 3. (d) Detaches at the termination site.

26. Transcription Bubble

27. Transcription in Prokaryotes (Cntd.) 2. Promoter A short sequence where RNA pol binds eg. TTGACA. Is not transcribed. Variable efficiency. 3. Elongation No primer. First nucleotide usually ATP or GTP. Transcription bubble moves ~ 50 nts/sec.

28. Transcription Initiation

29. Transcription (Cntd.) 4. Termination At the end of the gene “stop” sequences cause transcription to stop, the RNA-DNA hybrid to dissociate, & the DNA in the transcription bubble to rewind. Example of a Stop Signal: Series of GC base pairs, followed by series of AT base pairs  GC hairpin followed by a polyU sequence. Hairpin causes RNA pol to pause & the pairing of U with A is weak  Dissociation.