BIO 240 October 10 2014 RNA II RNA II RNA structure The discovery of mRNA Synthesis of RNA Transcription RNA polymerase Promoters Termination RNA modification Capping Polyadenylation Splicing RNA degradation Overview The relationship between transcription and translation Translation Types of RNA Evidence for mRNA Synthesis of a specific type of RNA in phageinfected cells E coli bacteria Inoculation with bacteriophage T2 Lytic cycle begins Injection of DNA into host cell Synthesis of RNA Protein synthesis Label culture with radioactive P 32P for a short period pulse chase Extract nucleic acids RNA Digest into ribonucleotides phosphate sugar base Separate nucleotides and measure radioisotope label in A C G and U Base G A U C Base composition of 32P RNA E coli only T2 inoculated 0 94 0 97 1 11 1 00 1 00 1 60 1 66 1 00 Base Base composition of DNA E Coli T2 phage G A T C 1 20 0 80 0 80 1 20 1 00 1 65 1 65 1 00 Astrachan Volkin 1956 1960 RNA synthesis Transcription DNA NTPs N P P P R Nucleosidetriphosphate DNA RNA pyrophosphate PPi DNA 3 OH D P D P D P D P D P D P D P D P D P D P 5 A C G T G T G C A G Hydrogen bonds U G C A C A C G P R 3 OH 5 P R P R P R P R P R P R P R 3 OH RNA G PPi PPP R template Transcription 5 3 Coding strand Q Similarities between RNA and DNA synthesis Differences Comparison of DNA synthesis and RNA synthesis DNA Replication RNA Transcription Requires a template Proceeds in 5 to 3 direction Energy from dNTP cleavage Same most of the time Same from NTP cleavage Requires a primer Proofreading Both strands NO primer LITTLE proofreading One strand at a time Transcription Nucleotide addition by RNA Polymerase II RNA DNA 3 OH Mg NTP Tri phosphate RNA POLYMERASE II purple yellow Kornberg PNAS 104 12955 12961 2007 Roger Kornberg Nobel Prize Winner 2006 RNA Polymerase Electron microscopic image of genes being transcribed Transcription of ribosomal RNA Amphibian oocyte Note rRNA genes reside in a cluster s of many copies nucleolar organizer region NOR Overview The relationship between transcription and translation Translation Types of RNA Transcription initiates at the promoter of the gene Case I Prokaryotes 3 UTR CDS Terminator RNA polymerase Case I Prokaryotes Sigma factor RNA Pol requires a sigma factor to recognize a promoter Sigma factor binds transiently Different Sigma factors target the polymerase to different promoters gene regulation Prokaryotic RNA polymerase continued c Helicase activity d Basepairing Q How to prove that the stem loop serves as a terminator Transcription in Eukaryotes Case II Eukaryotes Three RNA polymerases Pol I most ribosomal RNAs Pol II all messenger RNAs Pol III transfer RNAs 5S rRNA and snRNAs more Transcription initiation occurs at promoters Case II Eukaryotes The roles of general transcription factors TF II s RNA polymerase II Transcription bubble RNA polymerase II No sigma factor How to make your own RNA at home RNA Assembly Kit 2 1 TBP binds to the TATA box in the minor groove of the DNA and bends the DNA by 80o Kornberg PNAS 104 12955 12961 2007 RNA II RNA structure The discovery of mRNA Synthesis of RNA Transcription RNA modification mRNAs in eukaryotes Capping Polyadenylation Splicing RNA Degradation does not apply in prokaryotes mRNA modification I G P P P5 5 P N N mRNA3 3 G P P N N mRNA 3 Capped mRNA Why cap mRNAs Stabilizes RNA and boosts translation initiation mRNA modification II Polyadenylation 5 The 3 end of the mRNA is extended by a tail of 100 A s Poly A polymerase template independent Translational efficiency mRNA stability AAAA100 300 mRNA modification III Many eukaryotic transcripts contain introns which are spliced out Primary Transcript mRNA 3 5 Cap 5 Cap AAAAAA Intron Exon 1 Exon 2 AAAAAA Mature mRNA 5 Cap AUG Stop 5 Untranslated region Open reading frame ORF 5 UTR Coding Sequence CDS AAAAAA 3 Untranslated region 3 UTR Splicing occurs co transcriptionally RNA Polymerase II Exon Intron Spliceosome INTRONS are spliced OUT How are introns recognized Intron Consensus sequence G U A G Intron Exon junction The mechanics of intron splicing Overview Spliceosome proteins Small nuclear RNAs U1 U2 U4 U5 U6 The spliceosome is a ribozyme The mechanics of intron splicing Detail The mechanics of intron splicing Level III Self splicing introns Catalytic RNA Tom Cech and Sidney Altman 1981 RNA world Other ribozymes ribosome spliceosome mRNA modification PolII transcripts in eukaryotes Capping Polyadenylation Splicing Molecular biology Implications for gene function Alternative splicing yields multiple proteins from a single gene Splice site mutations can cause loss of function Implications for evolution Exon shuffling can create new genes Implications of mRNA splicing I Alternative splicing Multiple proteins from one gene Example Tropomyosin 5 splice site Splice donor site 3 splice site Splice acceptor site Implications of mRNA splicing II A mutation in a splice site commonly causes loss of function Example The m6 allele has a mutation in an intron exon junction splice site Implications of mRNA splicing II Allelic Series Example Mutant alleles in Phenylalanine hydroxylase Phenylketonuria gene Intron Mutations affecting the protein Exon Mutations affecting mRNA splicing Implications of mRNA splicing III New genes can arise by illegitimate recombination between introns exon shuffling Gene 1 Gene 2 Illegitimate recombination Chimeric gene The new gene combines functions of two separate genes into one RNA II RNA structure The discovery of mRNA Synthesis of RNA Transcription RNA modification Pol II transcripts in eukaryotes Capping Polyadenylation Splicing RNA degradation The fates of mRNA mRNA AAAA 5 Translation Degradation AAAA A Deadenylation Decapping endonuclease 3 5 5 3 exonucleases AAAA AAAA RNA interference The ABC A Trigger double stranded RNA e g virus RNA or short hairpin RNA Dicer endonuclease B Silencer miRNA siRNA 21 24 bases guide RNA mRNA 7mG C Target RISC endonuclease RNA degradation Slicer activity Translational repression Mello and Conte Nature 2004 RNAi I Sources of double stranded RNA Sources of dsRNA A Trigger dsRNA B Silencer 1 RNA Viruses 2 Endogenous transcripts Bidirectional transcription Inverted repeat genes 3 Exogenous RNA Microinjection Other Dicer endonuclease Biological role miRNA siRNA 21 24 bases Virus resistance plants Gene regulation development Control of transposable elements guide RNA Applications mRNA 7mG RISC endonuclease C Target Virus resistant plants Experimental tool RNA degradation Slicer