TRANSCRIPTIONAL PROCESSES: WHAT BASICALLY CONTROLS THE TRANSCRIPTIONAL EXPRESSION OF GENES, AND HOW ARE MRNA’S UTILIZED

1. TRANSCRIPTIONAL PROCESSES: WHAT BASICALLY CONTROLS THE TRANSCRIPTIONAL EXPRESSION OF GENES, AND HOW ARE MRNA’S UTILIZED

3. WHY DO WE CARE ABOUT KINETICS AND WHAT IS THE RELEVANCE TO DEVELOPMENT? • KINETIC METHODS PROVIDE ACCESS TO THE ACTUAL QUANTITIES OF THE PLAYERS (MRNA’S AND PROTEINS) • KINETIC METHODS TELL US ABOUT THE REAL-TIME PACE OF LIFE PROCESSES; THE REAL DIMENSIONS OF LIVING PARTS • KINETIC MODELS CAN, AND OCCASIONALLY DO, ILLUMINATE HOW A PROCESS WORKS • IN GENERAL: DEVELOPMENT IS A DYNAMIC PROCESS • SOME OF THE LOGIC OUTPUTS IN DEVELOPMENT ARE BOOLEAN, SOME (PARTICULARLY IN LATER AND POST EMBRYONIC DEVELOPMENT) CONTINUOUS; SIMILARLY, THE GENOMIC REGULATORY CODE AND NETWORK CONTROL SUBCIRCUIT TOPOLOGIES ARE DISCONTINUOUS, WHILE BIOCHEMICAL EXECUTION IS QUANTITATIVE AND CONTINUOUS.

4. Occupancy Occupied site Unoccupied site P PDs Ds Equilibrium constant

5. Occupied specific site Occupied nonspecific site Unoccupied specific site A A PDs PDN Ds DN Unoccupied nonspecific site 105

6. A B Double occupancy and cooperativity kRB kRA • Factors concentration, A(t), B(t). • Relative equilibrium constants (binding site affinities), kRA , kRB. • The interaction between the transcription factors increases the complex stability, cooperativity factor –

7. How do we compute occupancy given K equilibrium?How do we relate occupancy to transcription kinetics?

10. mRNA Synthesis rate B V A D RNAp RNAp RNAp RNAp RNAp RNAp S F RNAp High I rate Low I rate Initiation rate – Depends on the enhancer strength. The maximal rate is limited by the polymerase translocation rate. About 100 bp should be cleared before the next initiation can happen. At 15° the maximal initiation rate is 6 initiations/min. Polymerase translocation rate – Temperature dependent, sequence independent. At 15° it is 6-9 nt/sec. KT(T+10)/KT (T)=2 RNA processing – About 20 minutes, 5’cap+polyA done while still on the DNA. mRNA export rate – Fast (minutes, on average) not rate limiting. mRNA turnover rate – highly variable, sequence dependent (3’ UTR). 2 min – 20 hours. Average 2-3 hours.

11. Primary transcripts in amphibian oocytes Davidson EH, Gene Activity in early development, 3rd edition, academic press. Triturus Xenopus

12. Primary transcripts In sea urchin oocytes

13. Primary transcripts in mouse oocytes

14. Primary transcripts in rabbit oocytes

15. B A RNAp RNAp RNAp RNAp RNAp F mRNA Synthesis rate At 15°, at maximal speed 9 nt/sec, average gene size 30,000bp, How much time does it take to get the first mRNA molecule once transcription starts? Elongation: t = 30,000/9 =3333.3 sec ~ 1 hour. mRNA processing: 20 minutes. After this time the amount of mRNA depends on the initiation rate and on the mRNA turnover rate only (assuming that the RNA processing efficiency is 100%).

16. =3 Initiation/min mRNA Synthesis rate - initiation rate, 0-6 initiations/min. - mRNA turnover rate Solution: # mRNAmolecules/cell Time [min]

17. Protein synthesis rate Translation rate = translation initiation rate, limited by the ribosome translocation rate that is sequence independent, temperature dependent: At 15°, KT(15°) = 2 molecule protein/(min × molecule mRNA). Protein turnover rate – highly variable, from minutes to many days, sequence dependent. So just 10 mRNAs/cell can make 1200 molecules of protein in 1hr, enough for a good transcription factor to bind a sinificant fraction of target sites.

18. Protein synthesis rate Solution: # Proteinmolecules/cell # mRNAmolecules/cell Time [min] Time [min] =3 Initiation/min kt- 2 molecules/(min × mRNA molecule) kdP- Ln2/30 ~ 0.023 = Ln2/40 ~ 0.017

19. Protein Synthesis in Lytechinus pictus oocyte Davidson EH, Gene Activity in early development, 3rd edition, academic press.

20. dmRNA(t)/dt = ks – kd(t) ks can vary greatly; kd has a common default value. Occasional mRNAs have lower kd dProtein(t)/dt = kt.mRNA(t) – kpd.Protein(t); kt has a common default value per species and so does kpd. Occasional Proteins have lower kpd DEFAULT BOSYNTHESIS KINETICS

22. TEMPERATURE RESPONSE OF EMBRYONIC MACROMOLECULAR BIOLOGY - A GENERAL RULE IS RATE OF PROCESSES DEPENDS ON TEMPERATURE AS Q10=2 : RATE AT t+10/RATE AT t = ~2, WHETHER WE ARE LOOKING AT MORPHOGENETIC FUNCTIONS OR POLyMERASE TRANSLOCATION…FOR EXAMPLE LYTECHINUS AT 24c RUNS JUST ABOUT 2X THE RATE OF SP AT 15C; AND Drosophila pol rate is 20nt/sec at 22C while mammalian rate is 30-40nt/sec at 37Cfurthermore development is normal within allowable range at various ratesHOW CAN THIS BE?

24. Cascade behavior

26. STOCHASTICITY AND “NOISE” IN TRANSCRIPTION

27. protein molecules in a single cell mRNA molecules in a single cell Time (minutes after activation) Time (minutes after activation)

28. Number of protein molecules per cell averaged over 50 cells Number of mRNA molecules per cell averaged over 50 cells Time (minutes after activation) Time (minutes after activation)

29. Number of protein molecules per cell averaged over 200 cells Number of mRNA molecules per cell averaged over 200 cells Time (minutes after activation) Time (minutes after activation)