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6. Alternative splicing controls sex determination in Drosophila. Sexually dimorphic characteristics. Alternative splicing controls sex determination in Drosophila. Polyadenylation of the 3’ end of the nascent transcript is required for stability. Polyadenylation occurs in two phases: - PowerPoint PPT Presentation
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McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
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McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Alternative splicing controls sex determination in Drosophila
Sexually dimorphic characteristics
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Alternative splicing controls sex determination in Drosophila
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Polyadenylation of the 3’ end of the nascent transcript is required for
stability• Polyadenylation occurs in
two phases:
A slow phase mediated by Poly A Polymerase (PAP) during which approximately 12 A residues are added on to the cleaved 3’ end.
This structure is recognised by Poly A Binding Protein II (PABPII-to distinguish it from the cytoplasmic PABP), which catalyses the rapid addition of ~200 A residues.
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Splicing variants or isoforms provide added diversity: Alternative Splicing
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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Protein synthesis IProtein synthesis I
chapter 4chapter 4
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© R. Roy, 2010© R. Roy, 2010
How Is the Information Content in How Is the Information Content in DNA Transcribed to Produce RNA?DNA Transcribed to Produce RNA?
The genetic code: specifies which amino acids will be used to build a protein
Codon: a sequence of three bases. Each codon specifies a particular amino acid.
Start codon: AUG—initiation signal for translation (translational start site)
Stop codons: stops translation and polypeptide is released (translational termination signal)
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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The three roles of RNA in The three roles of RNA in protein synthesisprotein synthesis
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Figure 12.6 The Genetic Code
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Figure 12.8 Transfer RNA
anticodon
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Pre-tRNAs are transcribed by RNA Pol III and thereafter are processed to yield mature tRNA
• 1) the 5’ end sequence is removed
• 2) a short intron is removed
• 3) CCA is added on to the 3’ end
• 4) extensive modification of internal bases
Pre-tRNAs undergo critical processingPre-tRNAs undergo critical processing
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Pre-rRNA transcription units are Pre-rRNA transcription units are arranged in repetitive clustersarranged in repetitive clusters
Since rRNA accounts for approximately 80% of the total RNA in the cell, it has to be transcribed extensively to be able to satisfy the continuous demand
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Eukaryotic rRNA is transcribed in Eukaryotic rRNA is transcribed in units comprising the 18S, 5.8S and units comprising the 18S, 5.8S and
28S rRNAs28S rRNAs
the arrangement 5'-3' 18S - 5.8S - 28S is invariant within and between species
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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the pre-rRNA transcript is extensively processed in the nucleolus. Not only is it cleaved into its components, but also numerous bases are chemically modified.
all the pre-rRNA processing is executed through small nucleolar RNAs, which are part of snoRNPs.
unlike the other rRNAs encoded by the single pre-rRNA molecule, 5S rRNA is transcribed outside the nucleolus and arrives there by diffusion and is not further processed
Precursor rRNA is extensively Precursor rRNA is extensively processedprocessed
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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rRNA folds into conserved stem-rRNA folds into conserved stem-loop structuresloop structures
the overall stem-loop structure of ribosomal RNA is highly conserved among the thousands of species investigated to date.
(molecule shown is 16S bacterial rRNA, similar to 18S eukaryotic rRNA)
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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rRNA is an integral part of the rRNA is an integral part of the protein synthesizing machine, the protein synthesizing machine, the
ribosomeribosome
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Assembly of the pre-initiation complex
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Assembly of the pre-initiation complex
Protein synthesis can be negatively regulated by phosphorylation of eIF2.
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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RibosomesRibosomes
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The tRNAThe tRNAiiMetMet is needed to start is needed to start
synthesis of a polypeptide chainsynthesis of a polypeptide chaintwo types of tRNAs
exist for the methionine codon
both are charged by
the same aminoacyl
tRNA synthetase
tRNAMet is exclusively used for elongation of a polypeptide chain
tRNAiMet is exclusively
used for initiation of a polypeptide chain
the methionine in bacterial tRNAi
is modified by addition of a formyl group
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Assembly of the pre-initiation complex
Protein synthesis can be negatively regulated by phosphorylation of eIF2.
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
A 5' cap is required for the efficient A 5' cap is required for the efficient translational initiation of eukaryotic translational initiation of eukaryotic
mRNAsmRNAs Because the dimeric
guanyltransferase binds to the phosphorylated C-terminal domain (CTD) of RNA polymerase II only mRNA transcripts are capped!
therefore, only class II (mRNA) transcripts are efficiently translated!
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
• Sonenberg et al. purified a protein that bound the 7mGDP cap specifically using an affinity chromatography approach
• Addition of the purified protein to capped mRNAs increased the translational efficiency in vitro, but did not affect uncapped transcripts.
A cap-binding protein is A cap-binding protein is required for efficient required for efficient
translational initiationtranslational initiation
X
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
eIF4 is instrumental in recruiting eIF4 is instrumental in recruiting an mRNA to the preinitiation an mRNA to the preinitiation
complexcomplex next, a protein
complex named eIF4 binds the pre-initiation complex as well as the cap of an mRNA.
An associated helicase activity removes any secondary structure in the 5’ region of the transcript...
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
The initiation complex uses ATP energy and the helicase activity of eIF4A to scan the mRNA until the
initiation codon is found (usually the first AUG).
eIF4E-CapeIF4G-binds eIF3eIF4A-RNA helicase (ATP)
ACC G
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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eIF4 and mRNA are recruited to form an initiation complex
Activity of the cap-binding subunit eIF4E can also be regulated.
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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Bacterial translation initiation Bacterial translation initiation requires the Shine-Dalgarno boxrequires the Shine-Dalgarno box
the ribosome has to recognize the start codon of the ORF the ribosome start site for protein synthesis is directed toward the AUG
initiator codon by the Shine-Dalgarno box, a 5-8bp sequence that can base pair with the 16S rRNA molecule in the small ribosomal subunit
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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The Shine-Dalgarno box positions the The Shine-Dalgarno box positions the AUG initiation codon for pairing with AUG initiation codon for pairing with
the tRNAthe tRNAii the pairing of the Shine-Dalgarno box with the 16S rRNA results in a match of the initiation AUG codon with the initiator tRNA (tRNAi)
in mammalian cells a relatively conserved
sequence ACCAUGG recognises the start site (AUG)- KOZAK sequence (after Marilyn KOZAK)
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Recruitment of the large ribosomal subunit completes the
initiation phase.
Commitment to translation(can no longer dissociate)
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