Lecture'24'*'Chapters'38'&'39'Eukaryo8c'RNA'processing'and'the'gene8c'code'“Now%that%we%have%all%this%useful%informa3on,%it%would%be%nice%to%do%something%with%it.”%From%the%Unix%Programmer’s%Manual%%%%%The%RoseCa%Stone,%inscribed%in%196%B.C.%Outline'• Ribosomal'RNA'processing'• tRNA'processing'• mRNA'processing'• RNA'binding'proteins'• The'gene8c'code'• Wobble'hypothesis'• tRNA'structure'• tRNA'an8codon'binding'to'codon'• Aminoacyl*tRNA'synthetase'rRNA is made by Pol I in the Nucleolus • rRNA genes are arranged as tandem repeats. • In humans, there are approximately 400 rRNA genes in 5 clusters (on different chromsomes). • Localized transcription and processing take place in the nucleolus. rRNA Gene rRNA Gene rRNA Gene Transcribed Spacer Transcribed Spacer Etcà ßEtcThe'processing'of'eukaryo8c'pre*rRNA'rRNA Processing is NOT SplicingWhat does “S” mean? “Svedberg Units”Sedimentation Coefficient The functional ribosome is the 70S which is composed of one 30S subunit and one 50S subunit. 40S' 60S' 80S' In'eukaryotes'Ac8ve'form'Eukaryo8c'Ribosomal'RNA'(rRNA)'4.22'x'106D'ribosome'30S'50S'70S'prokaryotes'/16S'/23S'+'5S'/21' /34'Transfer'RNA'precursor'processing'Trailer'tRNA contains modified bases. Two modified Uʼs found in tRNA. There are many other modified bases (over 100 known). Modifications are introduced after the RNA is transcribed (post-transcriptionally). Ψ"U DEukaryo8c'mRNA'Processing'Pre*mRNA'mRNA'Eukaryo8c'mRNA'Ini8a8on'codon'Monocistronic'Coding'Region'Poly(A)'tail'5’Cap'5’'Untranslated'Region'3’'Untranslated'Region''7*methylguanylate'(m7G)'H'H'Caps'0'Capping'the'5ʹ′'end'Capping'the'5ʹ′'end''7*methylguanylate'(m7G)'H'Caps'1'Capping'the'5ʹ′'end''7*methylguanylate'(m7G)'Caps'2'5ʼ mRNA Capping • Occurs immediately after the 5ʼ end is made – before transcription is completed. • Functions – Important for splicing – Protection from RNases – Increased stability – Enhances efficiency of Translation – Cap Binding Protein – Required for efficient transport from the nucleusPolyadenyla8on'of'a'primary'transcript.'PABP'='polyA*binding'protein'Poly(A) tails Most'eukaryo8c'mature'mRNAs'have'~100*200'adenine'residues'a^ached'at'their'3’*'end,'called'the'3ʹ′*poly'A'tail''• Functions • Nuclear'export'• Transla8on'• Stability'of'mRNA.Prokaryo8c'mRNA'Polycistronic'mRNA'DNA'Protein'A'Protein'B'Protein'c'RBS'RBS'RBS''Biochemistry:'The'Molecular'Basis'of'Life,'5/e''Alternate'Splicing'of'Monocistronic'pre*'mRNAs'Monocistronic:'only'one'coding'region'Released'exon'not'translated'but'degraded'Released'exon'not'translated'but'degraded'+'+'Protein'A’'Protein'A’’'mRNA'A’'mRNA'A’’'Alternative Splicing • The human genome contains only about 20,000 genes. • The average gene has 7 introns. • It is estimated that 95-100% of these genes produce 2 or more different mRNAs as a result of alternative splicing. • Some genes have enough introns that they could theoretically produce over 30,000 different proteins. Thatʼs more proteins from 1 gene than there are genes.Splice'sites'Invariant'in'eukaryotes'Polypyrimidine'tract'5’*'*3’'Phosphoester'bond'being'a^acked'New'Phosphoester'bond'alcohol'alcohol'ester'ester'The Splicing Reaction Exon'2'Exon'2'Exon'1'Exon'1'1st'transesterifica8on'2nd'transesterifica8on'Small'nuclear'ribonucleoproteins'(snRNPs)'Splicing'–'part'1'Splicing'–'part'2:''+'U2'and'U6'snRNAs'form'the'cataly8c'center'of'splicesomes''5’è'2’'phosphodiester'bond'Transcrip8on'and'processing'of'mRNA'are'coupled'1'2'3'4'RNA'edi8ng'liver'Small'intes8ne'h^p://www.sciencephoto.com/media/210444/enlarge'Ribosomal'subunits'cytoplasm'nucleus'Nuclear'pore'channels'Nucleocytoplasmic'transport'of'ribosomal'subunits'A set of bound proteins marks the RNA as ready for transport Nuclear'Export'Fully Processed mRNA is Specifically Transported to the CytoplasmRNA*binding'proteins'have'mul8ple'func8ons'• Regulate'post*transcrip8onal'processing'• Prevent'degrada8on'• Target'for'nuclear'export'• Cytoplasmic''targe8ng'to'site'of'transla8on'• Transla8onal'regulators'The Genetic Code • The genetic code relates the sequence of bases in mRNA to the sequence of amino acids in proteins. • Problem: There are 20 amino acids but only 4 bases. • So how do you specify 20 amino acids with 4 bases? – 1 nucleotide / amino acid can code for 4 amino acids. – 2 nucleotides / amino acid can code for 42 = 16 amino acids. – 3 nucleotides / amino acid can code for 43 = 64 amino acids. – So there must be at least 3 nucleotides / codon. The code is a triplet code: a 3 base codon specifies each amino acid.The'Gene8c'Code'codons'Characteris8cs'of'the'Gene8c'Code'1. Triplet:'20'AA'and'64'codons'2. Non*overlapping'3. Contains'no'punctua8on'( except'for'Start'and 'Stop )'4. Has'direc8onality.''The'code'is'read'in'the'5’'to'3’'direc8on'5. Degenerate'–'All'but'two'amino'acids'(Met,'Trp)'have'more'than'one'codon.'6. Nearly'Universal'mRNA is “read out” in 3 nucleotide units – codons. Each codon specifies either a particular amino acid or a translational stop. There are 3 possible reading frames in every message. Only one of them makes “sense”. The reading frame is set by the translation initiation process. 5’'3’'Evidence'for'the'Code'• Homopolymers:''UUUUUUU…*>'poly*Phe'''AAAAAAAA…*>''poly*Lys'''CCCCCCCCC…*>''poly*Pro''• Ordered'co*polymers:'– ACACACACACACAC…'*>'Thr*His*Thr*His…'Nirenburg'and'Ma^aei,'1961'The'gene8c'code'is'degenerate'codons'N’*'*C’'+1'Frequency'of'codon'usage'varies'What'translates'the'nucleo8de'code'into'amino'acid'code?'Aminoacylated'tRNAs'Reading'frame'An8codon'codon'Glu'The'general'structure'of'transfer'RNA'molecule'Amino'acid'a^achment'site'Acceptor'stem'An8codon'loop'DHU'loop'TψC'loop'Phosphorylated'5’'terminus'“Extra'arm”'(variable)'L3shaped'3°'structure''Cloverleaf3like'2°'structure'''Base'pairing'between'codon'and'an8codon'1st 2nd 3rd 3rd 2nd 1stThe'Code'is'Degenerate'• There'are'61'codons'for'inser8on'of'20''amino'acids'into'proteins,'but'there'are'far'fewer'types'of'tRNA,'46'in%E.%coli.'• Therefore'a'tRNA'must'have'an8codons'that'can'recognize'more'than'one'codon.'•
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