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 Etc rRNA Gene rRNA Gene rRNA Gene Transcribed Spacer Transcribed Spacer Etc In humans there are approximately 400 rRNA genes in 5 clusters on different chromsomes Localized transcription and processing take place in the nucleolus The processing of eukaryo8c pre rRNA rRNA Processing is NOT Splicing What does S mean Svedberg Units Sedimentation Coefficient Ac8ve form 40S 60S 80S In eukaryotes The functional ribosome is the 70S which is composed of one 30S subunit and one 50S subunit Eukaryo8c Ribosomal RNA rRNA ribosome 30S prokaryotes 50S 70S 4 22 x 106D 16S 21 23S 5S 34 Transfer RNA precursor processing Trailer tRNA contains modified bases U D Two modified U s found in tRNA There are many other modified bases over 100 known Modifications are introduced after the RNA is transcribed post transcriptionally Eukaryo8c mRNA Processing Pre mRNA mRNA Eukaryo8c mRNA 3 Untranslated Region 5 Untranslated Region Coding Region 5 Cap Ini8a8on codon Monocistronic Poly A tail Capping the 5 end 7 methylguanylate m7G Caps 0 H H Capping the 5 end 7 methylguanylate m7G Caps 1 H Capping the 5 end 7 methylguanylate m7G Caps 2 5 mRNA Capping Occurs immediately after the 5 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 nucleus Polyadenyla8on 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 3 poly A tail Functions Nuclear export Transla8on Stability of mRNA Prokaryo8c mRNA DNA RBS RBS RBS Protein B Protein A Polycistronic mRNA Protein c Alternate Splicing of Monocistronic pre mRNAs Released exon not translated but degraded Released exon not translated but degraded mRNA A Monocistronic only one coding region Protein A Biochemistry The Molecular Basis of Life 5 e mRNA A Protein 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 That s more proteins from 1 gene than there are genes Splice sites 5 3 Invariant in eukaryotes Polypyrimidine tract Phosphoester bond being a acked ester ester alcohol alcohol New Phosphoester bond The Splicing Reaction 1st transesteri ca8on Exon 2 Exon 1 2nd transesteri ca8on Exon 1 Exon 2 Small nuclear ribonucleoproteins snRNPs Splicing part 1 Splicing part 2 5 2 phosphodiester bond U2 and U6 snRNAs form the cataly8c center of splicesomes Transcrip8on and processing of mRNA are coupled 1 2 3 4 RNA edi8ng liver Small intes8ne Nucleocytoplasmic transport of ribosomal subunits cytoplasm Nuclear pore channels Ribosomal subunits nucleus h p www sciencephoto com media 210444 enlarge Nuclear Export A set of bound proteins marks the RNA as ready for transport Fully Processed mRNA is Specifically Transported to the Cytoplasm RNA 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 5 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 3 Evidence for the Code Nirenburg and Ma aei 1961 Homopolymers UUUUUUU poly Phe AAAAAAAA poly Lys CCCCCCCCC poly Pro Ordered co polymers ACACACACACACAC Thr His Thr His The gene8c code is degenerate codons 1 Frequency of codon usage varies What translates the nucleo8de code into amino acid code Reading frame codon An8codon Glu Aminoacylated tRNAs The general structure of transfer RNA molecule Amino acid a achment site Phosphorylated 5 terminus DHU loop Acceptor stem T C loop Extra arm variable Cloverleaf like 2 structure An8codon loop L shaped 3 structure Base pairing between codon and an8codon 3rd 2nd 1st 1st 2nd 3rd The 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 This led Francis Crick to propose the so called Wobble Hypothesis Wobble Hypothesis The rules for base pairing with the 3rd base in the codon are less strict than for the 1st and 2nd bases The 1st and 2nd bases of the codon pair with tRNA according to strict Watson Crick rules The 3rd base of the codon has a more relaxed set of rules This is wobble Wobble hypothesis accounts for degeneracy of the gene8c code Wobble base pairing G U An8codon Codon U G U A 5 G C U 3 5 G C G 3 Modified Bases in tRNAs Include Inosine in the Wobble Position Inosine Adenosine Inosine change H bond donor NH2 to acceptor C O Wobble hypothesis accounts for degeneracy of the gene8c code Wobble base pairing A An8codon I U Codon C I 5 G C U 3 or 5 G C A 3 The general structure of transfer RNA molecule Amino
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