Quantitative Molecular Biology

Transcription

1 Quantitative Molecular Biology PHYS 176/276 Instructor: Terry Hwa Winter 2018 What is quantitative biology? èquantitative biology biology + numbers/equations biology-inspired physics application of existing methods to bio problems è use numbers as clutches to gain predictive understanding Why quantitative biology? -- because biology is quantitative -- needed to formulate and test falsifiable predictions -- demanded by synthetic biology Role of theory link across different scales, i.e., from components to systems formulate expectation and predictions (via quantitative model) guide the design of new experiments and technology power: the generality of (falsifiable) ideas, not necessarily math cost : the simplifying assumptions, not necessarily forced by math, but required in order to reveal principles è This course: quantitative molecular biology of bacteria 1

2 Overview Review of molecular microbiology 1. biochemical aspect 2. mechanistic aspect 3. regulatory aspect 4. genomic aspect 5. quantitative/physical aspect 6. comparison to eukaryotes Systems biology 1. scope and focus 2. circuit as system-level descriptor 3. scope of this course v life of a bacterium: matter + energy è biomass chemical composition of biomass: CH 1.80 O 0.43 N (+ S, P, Mg, Fe, ) algae (photosynthesis): CO 2 + H 2 O + N 2 + hν è biomass + O 2 E. coli (minimal medium): glucose + NH 3 è biomass molecular species % dry wt protein 55 RNA 20 DNA 3 small molecules 3 lipid membrane 9 cell wall 10 energy cost (J/g) molecular composition: [total weight: g per cell; 70% water] energy cost of biosynthesis: [minimal medium: ~ 560 J/g dr. wt.] (cf latent heat of melting H 2 O: 334 J/g) 2

3 v metabolism sequester & breakdown nutrients derive energy generate carbon precursors sequester N, S, P, metals glucose (6C) biosynthesis amino acid nucleic acid lipids co-enzymes IlvBN, IlvGM, IlvIH isozymes glycolysis 4x ATP 2x pyruvate CO 2 H +, NADPH IlvC 2x pyruvate (3C) NADP + respiration fermentation 8x ATP NH 3 6x CO 2 (need O 2 ) 32x ATP 2x acetate + 2x CO 2 akg Glu H 2 O IlvD è but many organisms use fermentation even with oxygen (Crabtree effect); why? valine IlvE v metabolism sequester & breakdown nutrients derive energy generate carbon precursors sequester N, S, P, metals biosynthesis amino acid nucleic acid lipids co-enzymes degradation/recycling typical biochemical reaction: S + Cb Sb + C S: substrate b: component (e.g., CH 3, NH 2, e - ) C: co-enzyme (needed for difficult reactions) è most reactions catalyzed by enzymes (proteins) è flux of the products and by-products need to be balanced metabolic control via coordinated regulation of enzyme abundance/activity 3

4 feedback inhibition threonine NH 3 IlvA 1st reaction of pathway often inhibited by product same enzymes used for synthesis of valine and isoleucine must have enzymes responding differently to different products (isozymes) in E. coli K-12, ilvg is defective è valine sensitivity in minimal media è α-ketobutyrate toxicity (repressed by isoleucine) α-ketobutyrate IlvC IlvD IlvE pyruvate CO 2 IlvGM IlvBN H 2 O IlvC IlvD IlvE isoleucine CO 2 H 2 O valine pyruvate Overview Review of molecular microbiology 1. biochemical aspect 2. mechanistic aspect 3. regulatory aspect 4. genomic aspect 5. quantitative/physical aspect 6. comparison to eukaryotes Systems biology 1. scope and focus 2. circuit as system-level descriptor 3. scope of this course 4

5 v Central Dogma amino acids, NTP, dntp, lipids, sugar, NH 3, O 2 rpl DNA tsx rrna trna mrna ribosomes tsl ribosomal proteins structural proteins transporters enzymes srna regulators RNAp DNAp v DNA replication the four bases of DNA: pyrimidines (C, T) and purines (A, G) DNA synthesis 5

6 the replication fork initiation of DNA replication doubling time of E. coli can vary over 10x [ fastest doubling time: ~20 min] 40 min required to replicate chromosome fixed time of 20 min between completion of one round of replication and cell division è doubling time > 60 min: waiting time between division & replication è doubling time < 60 min: multiple replication forks è one replication origin every 1.7 μm (length of unit cell): fast growing cells are larger! Questions: - mechanism of replication initiation control? - how does the cell measure its volume? - effect of gene dose on protein level? 6

7 v transcription: DNA to RNA RNA trna DNA vs RNA: RNA synthesis: heavily transcribed genes coding ribosomal RNA transcriptional initiation elongation and termination 7

8 v translation: mrna to protein Overview Review of molecular microbiology 1. biochemical aspect 2. mechanistic aspect 3. regulatory aspect 4. genomic aspect 5. quantitative/physical aspect 6. comparison to eukaryotes Systems biology 1. scope and focus 2. circuit as system-level descriptor 3. scope of this course 8

9 v dependence on the environment: exponential growth: è bacteria can sense the adjust the environment and adjust its growth program according to nutrients provided by the medium coping with stressful conditions: motility: flagella synthesis and chemotaxis osmotic response: porin/channel synthesis heat shock response: chaperons quorum sensing, biofilms, bacterial community SOS response (e.g., to DNA damage) non-growth condition minimal medium rich medium energy cost (J/g) doubling time 2-3 hr 20 min stationary phase (E. coli can be dormant for > 10 years) sporulation (e.g., B. subtilis) competence, conjugation (exchange of genetic materials) need regulation to maintain optimal growth v Central dogma + regulation amino acids, NTP, dntp, lipids, sugar, NH 3, O 2 rpl DNA tsx rrna trna mrna ribosomes tsl ribosomal proteins structural proteins transporters enzymes srna regulators RNAp DNAp tsx initiation control by transcription factors (TF) tsl initiation control by srna and RNA-binding proteins tsx termination control by srna and anti-terminators control of mrna and protein degradation coupled to environmental signals; coord growth program 9

10 v transcriptional initiation control modulation of RNAp-promoter affinity via activators and repressors allosteric activation of RNAp è net result: rate of tsx init dependent on cellular conc of activators/repressors controlled by, e.g., inducer molecules v two-component signaling systems L S K periplasm cytoplasm PO 3 R D promoter gene 10

11 sporulation and competence control in B. subtilus [A. Grossman, 95] warnings: 1. control circuit often incomplete; 2. annotated links may be indirect quorum sensing: inter-cellular communication density-dependent gene expression can detect multiple signaling molecules potential for complex language 11

12 Overview Review of molecular microbiology 1. biochemical aspect 2. mechanistic aspect 3. regulatory aspect 4. genomic aspect 5. quantitative/physical aspect 6. comparison to eukaryotes Systems biology 1. scope and focus 2. circuit as system-level descriptor 3. scope of this course proteins: amino acid chain; typically ~300 a.a. in length each protein encoded by a gene ( ~1000 bases) bacterial genomes: circular chromosome(s) E. coli K-12 MG1655: 4.6Mb, ~4400 genes; 2/3 identified 4 different strains of E. coli: Mb (common: 3.8Mb) range for 150+ bacteria: Mb plant bacteria obligate intracellular parasites E. coli, B. subtilis, 12

13 approx 85% of the E. coli genome codes for proteins operon structure genes coding for proteins that function in the same pathway may be located adjacent to one another and controlled as a single unit that is transcribed into a polycistronic mrna called operon txn initiation control open reading frame txn terminator Question: origin of the operon organization? regulator of an operon is often located nearby (and divergently transcribed) different operons involved in the same function can be located far away other genomic elements and features transfer RNA: 61 different types, nt in length è codons not equally represented: 86 trna genes in E. coli MG1655 è codons not equally used in coding sequences [= codon bias] (strong bias especially in genes involved in transcription and translation) 13

14 other genomic elements and features ribosomal RNA: 5S, 120 nt; 16S, 1542 nt; 23S, 2904 nt (22 genes encoding rrna in MG1655) transfer RNA: 61 different types, nt in length small (regulatory) RNA: a few hundreds discovered so far prophages: ~10 (5,000 ~ 50,000 nt) insertion sequences (transposons) replication origin (oric) termination of replication (terc) plasmids: types: plasmids F, P1 R1 psc101 ColE1 copy # ~10 ~30 length 90 kb 102 kb 6.5 kb 7.2 kb compatibility: plasmids whose replication origins are regulated by the same control system are incompatible 14

15 Overview Review of molecular microbiology 1. biochemical aspect 2. mechanistic aspect 3. regulatory aspect 4. genomic aspect 5. quantitative/physical aspect 6. comparison to eukaryotes Systems biology 1. scope and focus 2. circuit as system-level descriptor 3. scope of this course dimensions DNA: 2 nm x 2 nm x 3.4 nm/turn small proteins: (few nm) 3 or ~10nt protein complexes, (10-20 nm) 3 or 30 ~ 60nt cell size: 1 μm 2 x 3 μm concentration: 1 molecule/cell ~ 1nM intracellular diffusitivity of protein: ~10 μm 2 /sec 15

16 abundance ribosomes: ~ 20,000 (52 proteins each) RNAp ~ 1,000 (a few pct available) proteins: 2x10 6 (TF: 10 ~ 1,000 / type) mrna: small fraction of RNA 0.1 ~ 100 copies/cell; peaked at 2 ~ 3 copies / cell Warning: many of these numbers are dependent on growth condition! rates transcription: elongation ~40 nt/s translation: ~ 15 aa/s transcription-translation coupling: infrequently translated mrna cleaved mrna half-life: < 5 min protein half-life: from cell-doubling time (passive decay) down to a few min (active proteolysis) Question: which of these numbers are related? 16

17 Overview Review of molecular microbiology 1. biochemical aspect 2. mechanistic aspect 3. regulatory aspect 4. genomic aspect 5. quantitative/physical aspect 6. comparison to eukaryotes Systems biology 1. scope and focus 2. circuit as system-level descriptor 3. scope of this course v size and structure eukaryotes: cells ~1000x larger DNA in nucleus linear chromosomes haploids and diploids organelles vesicles microtuble network 17

18 v Chromosome duplication chromatin structure (regulated) cell cycle v RNA splicing and transport only RNA with appropriate proteins bound are selected for transport out of the nucleus 18

19 v genome and organization genome size Organism M. genitalium E. coli Yeast Human Rice Lilly organization (human) Genome length 0.5 Mb 4.5 Mb 12 Mb 3,000 Mb 500 Mb 90,000 Mb No. genes 500 4,000 6,000 35,000 50,000? multiple replication origins large intergenic separation: 3Gb/30,000 genes = 100kb (mostly transposable elements) v Gene regulation in eukaryotes control of transcriptional initiation direct activation by recruitment of RNAp activation/repression by modifying chromatin structure control of entry of regulators into nucleus control of RNA splicing (e.g., alternative splicing) localization of mrna control of mrna life-time control of mrna translation ubiquitination system to tag protein for degradation It s the regulation, stupid! 19

20 Overview Review of molecular microbiology 1. biochemical aspect 2. mechanistic aspect 3. regulatory aspect 4. genomic aspect 5. quantitative/physical aspect 6. comparison to eukaryotes Systems biology 1. scope and focus 2. circuit as system-level descriptor 3. scope of this course Systems biology 1. Scope and focus: Ø biological systems whose functions are derived from the interaction of many sub-components Ø ex: from macromolecluar assemblies to ecological communities Ø current focus: subcellular and cellular processes, e.g., genetic circuits, protein interaction networks [Davidson et al, 2002] Ø long-term goals: mapping out the complete wiring diagram of the cell quantitative, predictive computational model of the cell 20

21 2. Circuit diagram as system-level descriptor? circuit diagram supplemented by component parameters provides a concise quantitative description of the system circuit topology not necessarily predictive of system function; need to know the properties of the nodes components connectivity network complexity electronic circuits simple & well-characterized; many (~10 9 ); fast (10-9 sec) physical interconnect between well-insulated components (1~2 inputs per node) iterated cascades from complex network wiring genetic circuits heterogeneous, most rates unknown; few (~1000); slow ( >10 min) multiply-connected (1~10 inputs per node); regulation at all stages combinatorial signal integration from complex molecular control Experimental & Computational Approaches traditional mol bio: one gene, one process (e.g., A activates B) high throughput methods bioinformatic analysis many genes, one/few process(es) (e.g., genome-wide survey of gene exp) quantitative expts and modeling design and synthesis of biomolecluar systems one/few genes, multiple processes (e.g., txn init/term, post-tsl modification, degradation, small genetic circuits) quantitative model of individual nodes qualitative circuit diagram linking many nodes systems biology: many components, many processes (e.g., predictive modeling of the cell) 21

22 3. scope of this course focus on simple systems (bacterial gene regulation) role of theory, modeling, and computation multiple aspects (e.g., tsx initiation, post-tsx control, degradation) [vertical vs horizontal integration] emphasize on quantitative connections between molecular and physiological (functional) aspects power of functional constraints v course content molecular interactions: ligand-protein,protein-dna and protein-protein transcriptional initiation control: activation, repression, and combo post-transcriptional control: attenuation, termination, degradation modeling genetic circuits: bistability and oscillation stochastic gene expression and phenotype growth physiology and metabolic control 22