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

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Aubrey Greer
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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 -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 NOVEMBER 2,

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 NOVEMBER 2, 2006 ROBERT A. LUE 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 Only interacts with TAR in HIV RNA transcripts

HIV Tat enhances proviral transcription Mechanism of Tat activity differs from that of typical transcription activators RNAP II: CTD: CycT: Cdk9: Tat: RNA

Eukaryotic RNAs undergo significant modification a) Eukaryotic RNAs are processed to produce functional mrna b) RNA splicing increases the diversity of

2 HIV Tat enhances proviral transcription Mechanism of Tat activity differs from that of typical transcription activators RNAP II: CTD: CycT: Cdk9: Tat: RNA polymerase II C-terminal domain Cyclin T Cyclin-dependent kinase 9 HIV Transactivator RNA processing: the diversification of nucleic acid function 1. Eukaryotic RNAs undergo significant modification a) Eukaryotic RNAs are processed to produce functional mrna b) RNA splicing increases the diversity of eukaryotic RNAs c) Transport of RNA out of the nucleus is regulated 2. Controlling HIV protein expression by regulating RNA export Lecture Readings Alberts: pp

Genetic information is further diversified at the RNA

Bacteria and Eukaryotes Eukaryotic RNAs are processed to

events: (1) Addition of a 5 -Cap (2) Addition of a 3

3 Genetic information is further diversified at the RNA and protein levels Genes are organized differently in Bacteria and Eukaryotes Eukaryotic RNAs are processed to produce functional mrnas Three major RNA processing events: (1) Addition of a 5 -Cap (2) Addition of a 3 -poly(a) tail (Polyadenylation) (3) Removal of Introns (RNA Splicing)

4 Nucleotide sequences determine intron boundaries R: A or G Y: C or U Three nucleotide sequences are required for splicing: 5 splice junction 3 splice junction Branchpoint adenosine Intervening intron sequence can range in length from 40 to 50,000 bases Splicing removes introns as branched lariats The branchpoint adenosine attacks the 5 splice site, cleaving the sugar phosphate backbone The 5 end of the intron is covalently linked to the adenosine, forming a loop The free 3 -OH attacks the 3 splice site, ligating the exons together and releasing the intron lariat

Two chemical reactions remove the intron lariat 1st reaction 2 -OH of adenosine attacks the phosphate of the guanosine at the 5 splice site (donates e - ) Exchanges one bond for another 2nd reaction

5 Two chemical reactions remove the intron lariat 1st reaction 2 -OH of adenosine attacks the phosphate of the guanosine at the 5 splice site (donates e - ) Exchanges one bond for another 2nd reaction 3 -OH of the 5 exon attacks the phosphate of the guanosine at the 3 splice site After the intron is spliced out, the number of bonds is unchanged No energy consumed RNA splicing is executed by the Spliceosome complex The Spliceosome is a large assembly of discrete small nuclear ribonucleoprotein particles (snrnps), each made up of proteins and small nuclear RNAs Structural rearrangements within the Spliceosome depend on base-pairing between snrnas and the pre-mrna

RNA splicing is executed by the Spliceosome complex Many spliceosome components reside in discrete nuclear bodies called speckles The dynamic behavior of

Eukaryotic RNAs undergo significant modification 2.

6 RNA splicing is executed by the Spliceosome complex Many spliceosome components reside in discrete nuclear bodies called speckles The dynamic behavior of speckles is linked to transcription (activity of RNA Pol II) D. Spector RNA processing: the diversification of nucleic acid function 1. Eukaryotic RNAs undergo significant modification 2. Controlling HIV protein expression by regulating RNA export a) HIV Rev accelerates the nuclear export of selected viral RNAs b) Switching from early expression of regulatory proteins to the late expression of structural proteins and enzymes Lecture Readings Alberts: pp

7 HIV Rev: regulator of viral protein expression Essential for the control of HIV RNA splicing HIV RNAs exit the nucleus - Unspliced Single-spliced Double-spliced Rev enhances the amount of unspliced and single-spliced HIV RNA transcripts available in the cytoplasm for translation Nuclear export of HIV RNA Rev protein RRE Rev response element Early gene products Double-spliced RNAs produce viral regulatory proteins including Rev Late gene products Single & Unspliced RNAs produce structural and enzymatic components of HIV

HIV Rev mechanism of action XPO = Exportin Nuclear transport receptor that facilitates export through nuclear pores Ran = Protein that regulates XPO activity Rev coopts the XPO+Ran complex

8 HIV Rev mechanism of action XPO = Exportin Nuclear transport receptor that facilitates export through nuclear pores Ran = Protein that regulates XPO activity Rev coopts the XPO+Ran complex Translation: the RNA-directed synthesis of proteins 1. The role of RNA in protein synthesis a) Three classes of RNA are required to synthesize proteins b) mrnas are decoded in sets of three nucleotides c) The structure and function of transfer RNA d) Proofreading by aminoacyl-trna synthetase 2. The translation machinery and cycle Lecture Readings Alberts: pp McMurry: pp

Three classes of RNA are required to synthesize proteins Messenger RNA (mrna) serves as the informational template Transfer RNA (trna) are molecular adaptors that match amino acids to the

Decodes the mrna and promotes the polymerization of amino acids into proteins mrna sequences are decoded in sets of three nucleotides The Genetic Code Each nucleotide triplet in mrna is

9 Three classes of RNA are required to synthesize proteins Messenger RNA (mrna) serves as the informational template Transfer RNA (trna) are molecular adaptors that match amino acids to the mrna code Ribosomal RNA (rrna) associate with proteins to form the ribosome The ribosome is a macromolecular machine consisting of proteins and RNA Ribosome model with trna and rrna Decodes the mrna and promotes the polymerization of amino acids into proteins mrna sequences are decoded in sets of three nucleotides The Genetic Code Each nucleotide triplet in mrna is called a codon Codons are read consecutively 5 to 3 on the mrna Four nucleotides gives 4 3 or 64 possible codon triplets Most amino acids are encoded by several codons 3 codons encode a stop signal

10 mrna sequences can be decoded in three different reading frames mrna code can be translated in one of three reading frames Each reading frame is defined by the starting position of the first codon Each protein is translated in a specific reading frame Transfer RNAs match amino acids to codons 2 key domains Anticodon: Nucleotide triplet that base pairs with the complementary codon in mrna 3 -end: Attachment site for the appropriate amino acid Transfer RNA (trna) are molecular adaptors that connect specific amino acids with their matching codons Some trnas recognize more than one codon by tolerating mismatch base pairing at the 3rd position of the codon Some amino acids are matched by more than one trna

Aminoacyl-tRNA synthetase couples each trna with the appropriate amino acid 20 different aminoacyl trna

synthetase ensures that the correct amino acid is coupled with the correct trna Two sites (pockets) on

large Second, the editing site excludes the correct amino acid, but accepts and removes incorrect amino

11 Aminoacyl-tRNA synthetase couples each trna with the appropriate amino acid 20 different aminoacyl trna synthetases in eukaryotes Each recognizes one amino acid and all of its matching trnas Aminoacyl-tRNA synthetase ensures that the correct amino acid is coupled with the correct trna Two sites (pockets) on trna synthetase proofreads the amino acid First, the synthesis site excludes amino acids that are too large Second, the editing site excludes the correct amino acid, but accepts and removes incorrect amino acids that are similar in size Two step editing results in a low error rate of 1 in 40,000 trna couplings

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

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 NOVEMBER 2, 2006