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Alternative splicing controls sex determination in Drosophila
Sexually dimorphic characteristics
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Alternative splicing controls sex determination in Drosophila
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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.
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Splicing variants or isoforms provide added diversity: Alternative Splicing
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Protein synthesis IProtein synthesis I
chapter 4chapter 4
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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)
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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
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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
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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
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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
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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)
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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.
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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
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Assembly of the pre-initiation complex
Protein synthesis can be negatively regulated by phosphorylation of eIF2.
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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!
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• 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
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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...
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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
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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.
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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
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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)
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Recruitment of the large ribosomal subunit completes the
initiation phase.
Commitment to translation(can no longer dissociate)