1. Shine-Dalgarno Motif Ribosome binding site located about 13 bases upstream of AUG start codon SD sequence is: 5’-AGGAGGU-3’ Middle GGAG is more highly conserved Degree of match is positively correlated with translation rate

2. Assignment (due 9/9/04) Write perl code to detect Shine-Dalgarno motifs in E. coli DNA sequences. I will give you about 15 files, each has a single gene and one SD motif. (Ecoli.1, Ecoli.2, etc. in “examples”. You do not know which ORF to use (except that it is in the forward direction, so only 3 choices). S-D will be 6 bp (±5) upstream of ATG. Perfect matches are the exception, not the rule. Which ATG is a start codon? (Hint: gene will be at least 250 bp long, so if you encounter a stop codon early, it is not a gene. If you do not encounter a stop codon ever, it is not a gene.)

3. Eukaryotes (eucaryotes) Cells with a nucleus (us)

5. Gene expression: eukaryotes vs. prokayotes

7. RNA polymerase II promoters BRE - TFIIB recognition element TATA box INR - Intiator region Py.Py.A*.N.T/A.Py.Py (Py = pyramidine C/T) DPE - downstream promoter element Other elements 100-300 bp upstream CAT box and GC box

8. Initiation of transcription TFIIA, B, C … general transcription factors TBP - TATA binding protein Over 100 proteins involved TFIIH pulls DNA apart Then transcription factors are released so that transcription can occur Similar to sigma factor in prokaryotic transcription

9. In vivo is even more complex; note long distance regulatory proteins

10. Recognition sequences for most common splice (Y =C/U(T), R=A/G)

11. “Lariat” mechanism for removing intron

12. GT(U) - AG rule Most common splice mechanism U1, U2, U4, U5, U6 are snRNPs (small nuclear ribonucleoproteins) snRNPs are made up of snRNAs and proteins Assembly of snRNPs and other proteins is called a spliceosome

13. Alternative splicing!