BIOLOGY 305 1st Edition Lecture 18 Outline of Last Lecture I. Methods Continued (Identity Testing)II. Introduction to Replication and DiscoveryA. The Meselson-Stahl ExperimentIII. Mechanism of DNA ReplicationA. InitiationB. ElongationC. Termination IV. Vocabulary and Sample QuestionsOutline of Current Lecture I. Introduction to TranscriptionII. Mechanism of TranscriptionA. InitiationB. ElongationC. TerminationIII. RNA Processing (A Eukaryotic Feature)IV. Vocabulary and Sample QuestionsCurrent LectureI. Introduction to TranscriptionRecall once again: Essential features of genetic material- Diversity of structure, ability to replicate, mutability- Regulation is the fourth essentialGene expression is the success of:DNA (+replication)  transcription  RNA  translation  protein (+ prions) ( reverse transcription) (DNA  transcription factors)Recall the differences between DNA and RNA:- Uracil and 2’ OHRNA – between DNA and proteins in function and structure- can adopt unique structures and perform chemical functions (unlike DNA)has a Nucleotide sequence – which contains the genetic informationWhat is the difference between prokaryotes and eukaryotes?- Prokaryotes have no nucleus = transcription and translation happen in the same place- Eukaryotes have RNA processing and RNA transport in the nucleusII. Mechanism of TranscriptionGenes can be defined in more than one way, such as “unit of transcription”Basic Idea: a promoter is a piece of DNA that signals the RNA polymerase to bind to DNA (initiation) and elongates until it reaches a signal for termination (a termination sequence)A. Initiationa critical regulation step (elongation is mostly enzymatic)There are -35 and -10 sequences recognized by RNA polymerase (there is some conservation of elements but are not identical, there is a lot of variation)At the end of each RNA strand formed is usually a non-coding sequence called UTRsAt the 5’ end, there is usually a cap and on the 3’ end, there is usually a sequence of As’TATA box – eukaryotes have this promoter which function like -10, also facilitates unwinding double helix of DNAPoly-adenylation signal – often at the end of the sequenceExons – encoded, put into mRNA vs. Introns – non-encodedProkaryotic RNA polymerase holoenzyme – the pre-initiation complex- comprised of 5 subunits- The Sigma Unit:The sigma unit recognized the promoter and gives certain specificity to RNA polymeraseSigma factor is released when the mRNA begins transcribing- Others are transcription factors: there are 6 of themTFIID: is important for recognizing the TATA box, aka finds the promoterThe Initiation Complex:Sigma factor is released, TFIID stays to allow promoter ready for another polymerase to bind to that regionB. ElongationEukaryotic RNA Polymerases:RNA Pol I – transcribes ribosomal RNARNA Pol II – transcribes mRNA (has 14 subunits! Two subunits bind together = active site)RNA Pol III – transcribes tRNA some ribosomal DNA, and othersWhy three different RNA polymerases?There is so much ribosomal RNA, a specialized polymerase helps in not interfering with regulation of other kinds, response to metabolism of cellThere is also RNA IV and VC. TerminationImportant because it decides where the gene will endIn prokaryotes, there are two types of termination:1. Intrinsic – Rho independent: a hairpin loop forms on RNA, which weakens the interaction between RNA and DNA, thus the RNA just falls off2. Rho-dependent – relies on a special protein called Rho, which binds to short sequenceon RNA (called ‘Rho-binding site’), and induces terminationEukaryotes:1. Poly-adenylation signal – a specific short sequence that gets transcribed by polymerase but is then recognized by RNAase, which cuts the RNA on the inside (not outside like exonuclease)Poly A Polymerase – adds nucleotides to end of mRNA and creates a poly-A tailis different from RNA polymerase in that it is template-independentPoly-A tail – protects the 3’ end of RNA from degradationIII. RNA processing (a Eukaryotic feature)Primary RNA is processed into mature mRNA1) 5’ Capping2) Splicing and Alternative Splicing3) Poly-adenylationHappens co-transcriptionally (not sequential)coordinated by C-terminal Domain (CTD) – has several phosphorylation sites that reflect the state of the polymerase, acts as a binding site for other proteins, region on the end of RNA1. Formation of 5’ t-methylguanosine cap : like a backward GTP that protects (In red are the methyl groups)2. Splicing – extremely fast removal of intronsExons are later translated (but also contain UTRs), introns are intervening regionspre-mRNA – RNA that still has intronsMechanism:Splicing relies on 3 important DNA sequences: GU, A, and AGGU: splice donorA: branch pointAG: splice acceptorThe spliceosome is an snRNA + protein complex (snRNPs) surrounded by additional proteins that splices as such:1) Spliceosome is assembled2) 1st splicing reaction: cuts the donor site and attaches to branch sitespiled section forms a lariat structure, which eventually gets degraded3) 2nd splicing reaction: cuts the acceptor site and joins the two ends togetherexons are stable and do not get degradedAlternative splicing – can be regulated, some introns can be selected for inclusionThus, one gene can give rise to more than one polypeptide, one factor accounting for why there are ~100,000 protein variation for 25,000 genes in the human bodyAlternative Splicing:snoRNA  RNA processingsnRNA  spliceosometRNA  translationOne more feature of transcription:1) Prokaryotic have monocistromic or polycystromic RNA2) Eukaryotic cells have monocistromic RNA onlyIV. Vocabulary and Sample QuestionsUracil – the nitrogenous base in place of thymine, found in RNATemplate strand – the non-coding strand/anti-sense strand that is complementary to the RNA productCoding strand – non-template strand of DNAPromoter – the site to which RNA polymerase bindsRNA polymerase – the enzyme that transcribes DNA into RNA+1 – the first transcribed nucleotidecore RNA polymerase – the comlpex that binds to the promoteraccessory subunits - aid in binding and transcription initiationholoenzyme – the core enzyme together with accessory subunits closed promoter complex – during initiation, this is formed from the RNA polymerase holoenzyme binding to the promoteropen promoter complex – a short stretch of DNA unwound by enzymes