1a5’-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’

5’-UUA AUA UUC GAA AGC UGC AUC GAA AAC UGU GAA UCA-3’

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

ROBERT A. LUENOVEMBER 2, 2006

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: Transactivating regulatory protein

The Tat gene encodes an 86 - 104 amino acid transactivator protein Enhances the rate of viral replication up to 1000-fold

Mechanism of Tat activity differs from that of typical transcription activators

HIV Tat enhances proviral transcription

RNAP II: RNA polymerase II

CTD: C-terminal domain

CycT: Cyclin T

Cdk9: Cyclin-dependent kinase 9

Tat: HIV Transactivator

RNA processing: the diversification of nucleic acid function

1. Eukaryotic RNAs undergo significant modificationa) Eukaryotic RNAs are processed to produce functional mRNAb) RNA splicing increases the diversity of eukaryotic RNAsc) Transport of RNA out of the nucleus is regulated

2. Controlling HIV protein expression by regulating RNA export

Alberts: pp. 236-242

Lecture Readings

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)

Nucleotide sequences determine intron boundaries

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

R: A or G

Y: C or U

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

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

D. Spector

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)

RNA processing: the diversification of nucleic acid function

1. Eukaryotic RNAs undergo significant modification

2. Controlling HIV protein expression by regulating RNA exporta) HIV Rev accelerates the nuclear export of selected viral RNAsb) Switching from early expression of regulatory proteins to the

late expression of structural proteins and enzymes

Alberts: pp. 236-242

Lecture Readings

HIV Rev: regulator of viral protein expression

Essential for the control of HIV RNA splicing

HIV RNAs exit the nucleus -UnsplicedSingle-splicedDouble-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

Double-spliced RNAs produce viral regulatory proteins including Rev

Single & Unspliced RNAs produce structural and enzymatic components of HIV

Early gene products

Late gene products

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 synthesisa) Three classes of RNA are required to synthesize proteinsb) mRNAs are decoded in sets of three nucleotidesc) The structure and function of transfer RNAd) Proofreading by aminoacyl-tRNA synthetase

2. The translation machinery and cycle

Alberts: pp. 243-255McMurry: pp. 816-823

Lecture Readings

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

Decodes the mRNA and promotes the polymerization of amino acids into proteinsRibosome model with

tRNA and rRNA

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 43 or 64 possible codon triplets

Most amino acids are encoded by several codons

3 codons encode a stop signal

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

Transfer RNA (tRNA) are molecular adaptors that connect specific amino acids with their matching 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

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 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