RNA processing: the diversification of nucleic acid ...sites.fas.harvard.edu/~lsci1a/11-2.pdfRNA...
Transcript of RNA processing: the diversification of nucleic acid ...sites.fas.harvard.edu/~lsci1a/11-2.pdfRNA...
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