L I F E S C I E N C E S

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Tabitha Spencer
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1a L I F E S C I E N C E S 5 -UUA AUA UUC GAA AGC UGC AUC GAA AAC UGU GAA UCA-3 5

CTT TTG ACA CTT AGT-5 OCTOBER 31, 2006 ROBERT A.

Functional macromolecules and The directed synthesis of macromolecules Example of a

1 1a L I F E S C I E N C E S 5 -UUA AUA UUC GAA AGC UGC AUC GAA AAC UGU GAA UCA-3 5 -TTA ATA TTC GAA AGC TGC ATC GAA AAC TGT GAA TCA-3 3 -AAT TAT AAG CTT TCG ACG TAG CTT TTG ACA CTT AGT-5 OCTOBER 31, 2006 ROBERT A. LUE Cells have much to offer pathogens Cells contain essential resources: Functional macromolecules and The directed synthesis of macromolecules Example of a key protein function Protein monomers of Actin polymerize into filaments in the cytoplasm Key component of the cell cytoskeleton

Listeria: an example of protein cooption Example: Listeria monocytogenes

Bacterial protein (ActA) recruits actin monomers to one end of the

an example of protein cooption ActA coated microspheres Polystyrene

2 Listeria: an example of protein cooption Example: Listeria monocytogenes Bacteria that cause food poisoning Move in the cytosol of infected cells >10!m/min. Induces the polymerization of actin monomers at one end of the bacterium Bacterial protein (ActA) recruits actin monomers to one end of the bacterium Coopts existing cellular resources by direct binding Listeria: an example of protein cooption ActA coated microspheres Polystyrene microspheres (1!m dia.) are asymmetrically coated with ActA and incubated in a cytoplasmic extract Listeria-infected cell Lisa Cameron & Julie Theriot

Nucleic acids in action: Transcription and the programming of the cell 1. Sustained HIV replication destroys the human immune system 2. The principle of cellular cooption 3.

3 Nucleic acids in action: Transcription and the programming of the cell 1. Sustained HIV replication destroys the human immune system 2. The principle of cellular cooption 3. From DNA to RNA: Transcription a) Overview of intracellular information transfer b) RNA polymerase transcribes RNA from a DNA template c) Bacterial transcription as a model d) The starting and stopping of transcription in bacteria e) Eukaryotic transcription requires multiple initiation factors 4. HIV proviral transcription Lecture Readings Alberts: pp , McMurry: pp Overview of intracellular information transfer

Transcription of DNA into complementary RNA RNA polymerase

RNA Pol unwinds the DNA helix Catalyzes the polymerization of

formed The hybrid helix is disrupted to allow rewinding of

RNA Pol recognizes the promoter sequence RNA is synthesized

4 Transcription of DNA into complementary RNA RNA polymerase transcribes specific regions of DNA into complementary RNA RNA Pol unwinds the DNA helix Catalyzes the polymerization of RNA using DNA as a template A transient DNA-RNA helix is formed The hybrid helix is disrupted to allow rewinding of the DNA Bacterial RNA transcription Sigma factor subunit of RNA Pol recognizes the promoter sequence RNA is synthesized in the 5-3 direction Termination sequence halts RNA pol and induces release

Starting and Stopping transcription in bacteria Specific DNA sequences define the beginning and end of a transcriptional unit Promoter sequences are asymmetric -- serves to orient the RNA polymerase

5 Starting and Stopping transcription in bacteria Specific DNA sequences define the beginning and end of a transcriptional unit Promoter sequences are asymmetric -- serves to orient the RNA polymerase Regulatory proteins affect the rate of initiation at the promoter Starting and Stopping transcription in bacteria Terminator sequences induce the release the DNA template and RNA product The release process is facilitated by the formation of a RNA hairpin structure

transcribes genes encoding proteins RNA polymerase III transcribes genes encoding

6 Eukaryotic RNA polymerases Eukaryotic cells have three RNA polymerase enzymes Yeast RNA Pol II RNA polymerase I transcribes genes encoding ribosomal RNAs RNA polymerase II transcribes genes encoding proteins RNA polymerase III transcribes genes encoding transfer RNAs, ribosomal RNAs, and small structural RNAs Roger Kornberg Eukaryotic RNA polymerase II

Eukaryotic transcription requires multiple initiation factors Eukaryotic RNA polymerase II requires multiple transcription factors (TFs) for initiation General transcription factors assemble on all

II Addition of phosphate alters the activity of RNA Pol II, facilitating the completion of transcription Nucleic acids in action: Transcription and the programming of the cell 1.

7 Eukaryotic transcription requires multiple initiation factors Eukaryotic RNA polymerase II requires multiple transcription factors (TFs) for initiation General transcription factors assemble on all promoter sequences The TATA box (sequence) of the promoter binds the first TF Other TFs assemble at the promoter together with RNA Pol II A specific TF in the complex adds phosphate groups to RNA Pol II Addition of phosphate alters the activity of RNA Pol II, facilitating the completion of transcription Nucleic acids in action: Transcription and the programming of the cell 1. Sustained HIV replication destroys the human immune system 2. The principle of cellular cooption 3. From DNA to RNA: Transcription 4. HIV proviral transcription a) Reverse transcription b) Map of the HIV genome c) HIV Tat protein enhances proviral transcription d) Tat recruits host cell proteins that alter the activity of RNA polymerase II Lecture Readings Alberts: pp , McMurry: pp

to DNA (RNA-dependent DNA polymerase) 2) Digests RNA from RNA-DNA hybrid (RNAse

8 HIV Reverse Transcription HIV Reverse Transcriptase RT reverse transcribes the single-stranded RNA genome into double-stranded DNA before integration 1) RNA to DNA (RNA-dependent DNA polymerase) 2) Digests RNA from RNA-DNA hybrid (RNAse function) 3) Make DNA from DNA, i.e. double-stranded (DNA-dependent DNA polymerase)

9 Nucleoside analog RT inhibitors AZT is an analog of thymidine Lacks the 3 -OH required for the addition of the next nucleoside triphosphate Inhibits reverse transcription by competing for the nucleoside substrate and terminating chain polymerization Mechanism of AZT inhibition

10 Map of the HIV genome The HIV genome is 9,800 bases versus the ~ 3 billion base pairs of the human genome HIV structural gene products: Gag, Env Gag encodes matrix proteins; Env encodes envelope proteins HIV enzymatic gene products: Pol Pol protein is processed to yield three HIV enzymes Reverse transcriptase (copies viral RNA into DNA) Integrase (integrates viral DNA into the host cell genome) Protease (cleaves / processes specific viral proteins) Map of the HIV genome HIV regulatory gene products: Tat, Rev, Nef, Vif, Vpu, Vpr collectively + host cell factors, control the virus replication cycle Consist of two groups: 1) Proteins that are essential for HIV replication: Tat, Rev 2) Proteins that perform accessory functions which enhance replication and/or infectivity: Nef, Vif, Vpr, Vpu

HIV Tat: Transactivating regulatory protein The Tat gene encodes an 86-104 amino acid transactivator

sequence within the 5 LTR called TAR or Tat responsive element HIV Tat enhances proviral transcription

11 HIV Tat: Transactivating regulatory protein The Tat gene encodes an amino acid transactivator protein Enhances the rate of viral replication up to 1000-fold The protein interacts with a short sequence within the 5 LTR called TAR or Tat responsive element HIV Tat enhances proviral transcription RNAP II: CTD: CycT: Cdk9: Tat: RNA polymerase II C-terminal domain Cyclin T Cyclin-dependent kinase 9 HIV Transactivator

October 26, Lecture Readings. Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell

October 26, Lecture Readings. Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell October 26, 2006 Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell 1. Secretory pathway a. Formation of coated vesicles b. SNAREs and vesicle targeting 2. Membrane fusion a. SNAREs