1. Transcription BIT 220 Chapter 12 Basic process of Transcription Figures 12.3 Figure 12.5

2. Terminology Molecular Biology/Genetics: the study of gene structure and function at the molecular level Molecular Biotechnology: the ability to transfer specific units of genetic information from one organism to another Recombinant DNA Technology = gene splicing = genetic engineering =gene transplantation = gene cloning =molecular cloning

3. DNA Measured in base pairs (bp) kb - kilobases - 1000 bases 10.4 base pairs in one helical turn 3.4A (3.4 x 10 -4 um) -space between each base pair Molecular Weight of 1 NT pair 660 Genes are conventionally written with non-template (coding) strand on top in 5’-3’ direction Template in 3’-5’ direction

4. Genes Definition Linear sequence of DNA that contains all the necessary information for the production of a protein promoter – start codon—gene -- stop codon (ATG) (introns exons) TERMINOLOGY Express Gene - Transcribe/Translate MAKE PROTEIN Intron noncoding region of DNA Exon coding region of DNA Upstream 5’ end Downstream 3’ end ORF Open Reading Frame from start to stop codon

5. Figure 12.4

7. DNA TRANSCRIPTION Make single stranded complementary RNA strand from DNA Figure 12.7 (1) CODING STRAND of DNA= ‘identical’ to mRNA (exception uracil) called nontemplate strand (2) TEMPLATE Nucleotides complementary to DNA template are bonded to form mRNA (transcript, sense strand) RNA as intermediary - Figure 12.8 1. Ribose Sugar 2. Phosphate 3. Four Bases adenine guanine cytosine uracil

8. Figure 12.9 TRANSCRIPTION BUBBLE unwound DNA created by RNA polymerase site of transcription process Steps in Transcription A. Initiation B. Elongation C. Termination Figure 12.10

9. RNA Polymerase Protein 1 prokaryotes 3 in eukaryotes Holoenzyme (Complete Enzyme) 1.Core a. alpha recognizes upstream element b. Beta- binds ribonucleotides forms phosphodiester bonds c. Beta’ binds DNA template 2. Sigma - recognizes the core promoter elements (-35) directs polymerase to initiate transcription at a specific promoter

10. Promoter/Regulation Prokaryotes where RNA polymerase sits on DNA and begins transcription upstream of promoter (minus designation) downstream of promoter (plus designation) Prokaryotes a. -10 Box TATA box b. -35 box b. UP element Figure 12.11 Eukaryotes a. TATA box (-30) b. CAAT box (-80) c. GC box

11. Initiation 1. Polymerase sits on non-specific DNA 2. Sigma subunit searches and finds promoter 3. Polymerase binds to -35 region 3. B and B’ bind tightly to DNA 4. DNA begins melting at -10 region 5. Complementary nt are added and bonded at +1 6. Sigma subunit falls off polymerase Transcription occurs in 5’-3’ direction Asymmetrical Elongation - Figure 12.12

12. Termination of Transcription Figure 12.13 Intrinsic Terminators GC rich region in DNA Allows hairpin in RNA Cause polymerase to stall All subunits of polymerase fall apart mRNA is free DNA recoils

13. Transcription in Eukaryotes Promoters Transcription Factors Enhancers (100-1000 bp upstream) Transcription in Nucleus Translation in Cytoplasm 3 RNA Polymerases (Table 12.1)

14. Eukaryotic RNA Editing 1. 5’ capping Figure 12.19 7 methyl guanosine cap 2. polyA tail (3’) Figure 12.20 3. Introns removed - next slide FIGURE 12.5 and 12.15 Figure 12.17 for Initiation in Eukaryotes

15. Intron Splicing Figure 12.28 Most, but not all, eukaryotic genes contain introns. These introns are removed from mature mRNA They contain sequences which are often conserved. MECHANISMS (rRNA)Self Splicing (Autocatalytic Activity) intron excised via two phosphoester bonds (mRNA)Spliceosomes small nuclear RNA proteins (tRNA)Endonuclease and Ligase

18. Why use RNA? Protect DNA Allows control of proteins made RNA needs to move to new location