Lecture 24 Chapters 38 39 Eukaryotic RNA processing and the genetic code Now that we have all this useful information it would be nice to do something with it From the Unix Programmer s Manual The Rosetta Stone inscribed in 196 B C Outline Ribosomal RNA processing tRNA processing mRNA processing RNA binding proteins The genetic code Wobble hypothesis tRNA structure tRNA anticodon 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 eukaryotic pre rRNA rRNA Processing is NOT Splicing What does S mean Svedberg Units Sedimentation Coefficient Active form 40S 60S 80S In eukaryotes The functional ribosome is the 70S which is composed of one 30S subunit and one 50S subunit Eukaryotic 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 Eukaryotic mRNA Processing Pre mRNA mRNA Eukaryotic mRNA 3 Untranslated Region 5 Untranslated Region Coding Region 5 Cap Initiation codon Monocistronic Poly A tail Capping the 5 5 end 7 methylguanylate m7G Caps 0 H H Capping the 5 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 Polyadenylation of a primary transcript PABP polyAbinding protein Poly A tails Most eukaryotic mature mRNAs have 100 200 adenine residues attached at their 3 end called the 3 poly A tail Functions Nuclear export Translation Stability of mRNA Prokaryotic 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 Monocistronic only one coding region Released exon not translated but degraded Protein A Biochemistry The Molecular Basis of Life 5 e mRNA A 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 attacked ester ester alcohol alcohol New Phosphoester bond The Splicing Reaction 1st transesterification Exon 2 Exon 1 2nd transesterification Exon 1 Exon 2 Small nuclear ribonucleoproteins snRNPs Splicing part 1 Splicing part 2 5 2 phosphodiester bond U2 and U6 snRNAs form the catalytic center of splicesomes Transcription and processing of mRNA are coupled 1 2 3 4 RNA editing liver Small intestine Nucleocytoplasmic transport of ribosomal subunits cytoplasm Nuclear pore channels Ribosomal subunits nucleus http 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 multiple functions Regulate post transcriptional processing Prevent degradation Target for nuclear export Cytoplasmic targeting to site of translation Translational 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 Genetic Code codons Characteristics of the Genetic Code 1 Triplet 20 AA and 64 codons 1 Non overlapping 2 Contains no punctuation except for Start and Stop 3 Has directionality The code is read in the 5 to 3 direction 4 Degenerate All but two amino acids Met Trp have more than one codon 5 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 Mattaei 1961 The genetic code is degenerate codons ca p 1 Frequency of codon usage varies What translates the nucleotide code into amino acid code Reading frame codon Anticodon Glu Aminoacylated tRNAs The general structure of transfer RNA molecule Amino acid attachment site Phosphorylated 5 terminus DHU loop Acceptor stem T C loop Extra arm variable Cloverleaf like 2 structure Anticodon loop L shaped 3 structure Base pairing between codon and anticodon 3rd 2nd 1st 1st 2nd 3rd The Code is Degenerate There are 61 codons for insertion of 20 amino acids into proteins but there are far fewer types of tRNA 46 in E coli Therefore a tRNA must have anticodons 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 genetic code Wobble base pairing G U Anticodon U G Codon 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 genetic code Wobble base pairing A Anticodon I U Codon C I 5 G C U 3 or 5 G C A 3 The general structure of transfer RNA molecule Amino acid attachment site Phosphorylated 5 terminus DHU loop
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