1.Benjamin A. Pierce GENETICS A Conceptual Approach FOURTH EDITION CHAPTER 13 Transcription 2012 W. H. Freeman and Company

2. Chapter 13 Outline 13.1 RNA, Consisting of a Single Strand of Ribonucleotides,Participates in a Variety of Cellular Functions, 352 13.2 Transcription Is the Synthesis of an RNA Molecule from aDNA Template, 354 13.3 The Process of Bacterial Transcription Consists of Initiation,Elongation, and Termination, 359 13.4 Eukaryotic Transcription Is Similar to Bacterial Transcriptionbut Has Some Important Differences, 364 3. DNA TRANSCRIPTIONAsthma, cancer, heart disease, immune disorders and viral infections are seemingly disparate conditions. Yet they turn out to share a surprising feature. All arise to a great extent from overproduction orunderproduction of one or more proteins, the molecules that carry outmost reactions in the body. Sci. Amer. 1995 4. 13.1 RNA Consisting of a Single Strand of Ribonucleotides, Participates in a Variety of Cellular Functions The structure of RNA Primary structure Secondary structure 5. 13.2 Transcription Is the Synthesis of an RNA Molecule from a DNA Template The template: The transcribed strand: template strand. 6. 13.2 Transcription Is the Synthesis of an RNA Molecule from a DNA Template The transcription unit A promoter RNA-coding sequence Terminator 7. Transcription, mRNA processing, and translation Instructions Product DNA serves as the instruction manual for making proteins Going from instructions to product occurs in 3 main steps: 1. Transcription- The information contained in the DNA is copiedinto a complementary strand of RNA (ribonucleic acid)- This RNA copy is called messenger RNA or mRNA- Why is transcription necessary? (why copy the info)- Location issues- DNA is too valuable2. mRNA editing - The mRNA copy needs to be modified (cleaned-up) before it leaves the nucleus - Does not occur in prokaryotic cells3. Translation - Ribosomes read the mRNA and make a protein 8. Transcription Activation of transcription Transcription is gene specific - Each gene provides instructions for a single protein (usually) - When turned on, only that one gene is transcribed (unlike DNA rep) What tells a cell to start transcribing a specific gene?- Different signals for different genes in different cell types 1) Constituitive expression Some genes are transcribed continuouslyindependent of cell health or environmental conditions - Usually seen for those proteins that are needed 100% of the time - Example: ATP synthase, actin, tubulin2) Regulated expression - Allows for much tighter control of gene expression YYYPM- Signal from outside or inside the cellstarts the whole process- Often involves one or more signal transduction pathways 9. Prokaryotic transcriptionInitiation Finding the gene All genes (eukaryotic and prokaryotic) have a unique region of DNA sequenceupstream of (before) the transcriptional start site that serves as a binding site forthe RNA polymerase enzyme (makes RNA) - These regions are called promoterspromoterpromoter RNA pol General functions of promoters:1. Provides specificity Tells the cell where the gene of interest is - This is critical transcribing the wrong gene could be deadly!! - Each gene has a unique promoter sequence 2. Tell RNA polymerase where to start (they are usually at the beginning)- Analogy: Front cover of a book- No promoter (or mutated promoter), no transcription 3. Indicate which strand will be transcribed and the direction of transcription 10. Prokaryotic transcription Initiation Finding the gene Although each promoter is unique,most share a few common sequence elements called consensus sequences- These common elements are evolutionarily conserved- These common elements are extremely important for transcription - If mutated, transcription does not take place Prokaryotic promoters share 2 main types of consensus sequences - TATA box (aka Pribnow box) found 10 nucleotidesupstream from the start site (-10-15), sequence is usually TATAAT - TTGACA found 35 nucleotides upstream from start (-35). Exact sequence and spacing can vary!! RNA polymerase enzyme makes direct contactwith the promoter at these consensus sequences Unique seq. within and around promoters help determine rates of transcription 11. Prokaryotic transcriptionInitiation Binding of RNA polymerase to the promoter Prokaryotic cells only produce 1 type of RNA polymerase- Produces all of the mRNA, tRNA, and rRNA in the cell Prokaryotic RNA polymerase structure: - 2 alpha (), 1 beta (), 1 beta prime (), one omega ( ) and 1 sigma () subunit - , , subunits interact and form the CORE ENZYME - Together they have the basic catalytic function of producing new RNA - factor controls binding to the promoter (PROMOTER RECOGNITION) - After initial binding has been successful, the factor comes off the core- ANALOGY: factor is the general, the core enzyme is the troops - Bacteria make many different types of sigma factors- Different sigma factors recognize different promoter sequences- Examples: 70 controls transcription of general purpose genes54 controls transcription of genes only during nitrogen starvation 12. Concept Check 2What binds to the 10 consensus sequence foundin most bacterial promoters? a. The holoenzyme (core enzyme + sigma factor) b. The sigma factor alone c. The core enzyme alone d. mRNA 13. Concept Check 2What binds to the 10 consensus sequence foundin most bacterial promoters? a. The holoenzyme (core enzyme + sigma factor) b. The sigma factor alone c. The core enzyme alone d. mRNA 14. Prokaryotic transcriptionInitiation and elongation Once it binds to the specific promoter, RNA polymerase positions itself over the transcriptional start site- Chooses the start site based on its distance from the consensus sequences - No specific start signal exists RNA pol unwinds the DNA near the start site (transcription bubble created) RNA pol reads 1st ~5-10 DNA nucleotides and brings in complimentary RNAnucleotides (remember: U inserted across from A)- Connects them via phosphodiester bonds- Only transcribes one strand- Moves 53 (slowly initially) RNA polymerase changes shape after ~10 nucleotidesa) Cause it to lose its attraction for the consensus sequences - Allows it to begin moving along the geneb) Cause the sigma factor to fall off http://www.youtube.com/watch?v=WBcS3fKfbxs Once free, it moves quickly along the rest of the gene (53) 15. Prokaryotic transcription Termination RNA pol continues producing a complimentary RNA molecule until it transcribesa terminator signal (part of the sequence)- ANALOGY: Not like a stop light. More like spikes on the road Two major prokaryotic transcriptional terminator signals exist- Both must:a) Slow down the RNA polymeraseb) Weaken the interaction between the DNA and RNA in the bubble Properties of the two major prokaryotic terminator signals1. Rho-independent terminators - The DNA sequence contains an inverted repeat followed by a string of 6 adenines - Following their transcription, the inverted repeats formHydrogen bonds with each other within the RNA and form a hairpin loop - Loop formation causes the RNA pol to pause - The pause combined with weak hydrogen bonding between 6 straight A-U pairs cause the RNA to totally fall off of the DNA template 16. Prokaryotic transcription Termination ( two major prokaryotic terminator signals)2. Rho-dependent terminators - Also contain inverted repeats that cause hairpin loop formation in the new RNA - Slows down the polymerase- RNA contains a binding site for the Rho protein- Rho moves towards the 3 end- Catches up to the RNA polymerase and unzipsthe RNA from the DNA - Acts as a RNA/DNA helicase These do not contain adenine rich region after the inverted repeat Nearly all bacterial genes have one of these terminators 17. 13.4 Eukaryotic Transcription Is Similar toBacterial Transcription but Has SomeImportant Differences Transcription and nucleosome structure Chromatin modification before transcription. Promoters: Basal transcription apparatus Transcriptional activator proteins RNA polymerase II mRNA synthesis Core promoter TATA box TATAAAA, -25 to -30 bp, binded by transcription factors. 18. 13.4 Eukaryotic Transcription Is Similar toBacterial Transcription but Has SomeImportant Differences Transcription and nucleosome structure Chromatin modification before transcription. Promoters: Regulatory promoter Variety of different consensus sequences may befound in the regulatory promoters. Fig. 13.16 Enhancers: Polymerase I and polymerase III promoters. 19. Eukaryotic transcription Activation and Initiation Eukaryotic transcription follows the same basic steps of prokaryotic transcription- Activation, initiation, elongation, termination- Major differences exist in how they accomplish the above steps General features of transcriptional activation is similar in prokaryotic andeukaryotic cells- Some genes are constituitively expressed, others are regulated- The exact nature of the signals that activate transcription can be very different- Example: Adding testosterone to a bacterial cell would likely do nothing Initiation Making the DNA accessible- Eukaryotic DNA is tightly coiled around proteins calledhistones (and some nonhistone proteins)- DNA is negatively charged (phosphate groups), histones are very positively charged - Every 146 base pairs are wrapped around a histone octamer (8 histone proteins in a complex)- Each group is called a nucleosome 20. Eukaryotic transcriptionInitiation Making the DNA accessible Histones cont.- To initiate transcription, DNA in the area of the gene has to beloosened/unwound from histones and other proteins DNA is freed from histones in 2 major ways:1) Histone acetylation - Enzymes called histone acetyl transferases (HATs) add an acetyl group (CH3CO) to histones (nucleophylic attack)- This neutralizes their positive charge and causes them to lose their attraction for DNA- Occurs only in the area to be transcribed(it is specfic!) Deacetylases remove acetyl groups after transcription2) Chromatin remodeling - Some proteins move nucleosomes around (freeing up the DNA) without directly modifying histones 21. Interferon - JAK/STAT PathwayIFNJAK JAKSTAT 1PPPPY Y STAT 1STAT 1PP STAT 1 22. Interferon - JAK/STAT Pathway STAT 1 P PSTAT 1STAT 1 P Accessory TFP motifsTATAGAS motif STAT 1 23. Interferon - JAK/STAT PathwayAccessory TFfactorsSTAT 1 P Accessory TFP motifs TATAGAS motif STAT 1 24. Eukaryotic transcriptionInitiation Finding the gene (promoters and enhancers) Eukaryotic promoters- Much more complex than those found in bacteria- Some major consensus sequences (there are others): 1) TATA box - Very similar to the prokaryotic TATA box, except the sequence is slightly different (TATAAA) and it is located -25-30. 2) CAAT box - Located -70-80. Always contains either CAAT or CCAAT.-Mutation of this region usually significantly lowers rate of transcription3) GC box Usually has the sequence GGGCGG and is typically found~ -110 bps. Promoters can contain multiple copies of each of these consensus sequences The regions found between consensus sequences are unique in each promoter (see next slide) 25. Eukaryotic promoters- Sequences between and around the three common consensus sequences aredifferent in each promoter Notice that each promoter is unique. Allows the cell to recognize it anddistinguish it from other promoters/genes 26. Eukaryotic transcription Initiation Binding of RNA polymerases to the promoter Review of prokaryotic cells: A single type of RNA polymerase recognizes andbinds directly to consensus sequences in prokaryotic promoters Eukaryotic cells are a much more complicated they have multiple RNApolymerases and none of them recognize promoter sequences directly Eukaryotic cells have 3 RNA polymerases: 1) RNA polymerase I Produces ribosomal RNA (rRNA) 2) RNA polymerase II Produces messenger RNA (mRNA)- All mRNA is made by RNA pol II (what we will discuss) 3) RNA polymerase III Produces some types of rRNA and all tRNAs All function to transcribe a gene (DNA) into RNA (not just mRNA) All are much larger and more complex than the bacterial RNA polymerase- Needs to be more complex because eukaryotic gene diversity- Can have 20,000 different genes 27. Eukaryotic transcriptionInitiation Binding of RNA polymerases to the promoter If eukaryotic RNA polymerases do not recognize promoter sequences directly,how do they bind to the correct promoter? WITH THE HELP OF TRANSCRIPTION FACTORS! Transcription factors = Class of proteins that bind to DNA and help to recruitRNA polymerase enzymes to promoters(similar function as the __SIGMA_ in bacteria) - As a result, they help to regulate eukaryotic transcription - Two main classes 1) Basal transcription factors Common set of proteins needed to get transcription started (all promoters use these) give low levels of transcription2) Regulatory transcription factors Provide more specific transcriptional control Either activate or repress transcriptionabove/below basal levels 28. Eukaryotic transcription Initiation Binding of RNA polymerases to the promoter Binding of basal transcription factors to the promoter(and recruitment of RNA pol II):1) The TFIID complex binds to the TATA box through its TBP subunit- TBP = TATA-binding protein 2) This binding alters the shape of theDNA and allows for binding ofTFIIA and TFIIB- TFIIA = Stabilizes interaction betweenTBP and the DNA- TFIIB = Helps find the start site 3) RNA polymerase (escorted by TFIIF)then comes in and interacts with thepreassembled complexhttp://www.youtube.com/watch?v=JOBwqwxgJqc&feature=related 29. Eukaryotic transcriptionInitiation Binding of RNA polymerases to the promoter Binding of basal transcription factors to the promoter(and recruitment of RNA pol II):4) Other factors then come in and helpRNA pol II to gain direct access tothe promoter - TFIIH = Serves as a helicase to separate the strands during transcription- Once this giant complex of proteins isassembled on the TATA box, RNA pol IIwill leave most of the other proteinsbehind and start making mRNA- The above events occur during all eukaryoticgene transcription and provides justbasal levels of transcription - When a cell wants more or less than this, it will utilize regulatory transcription factors (discussed later) http://www.youtube.com/watch?v=gZtGrsr8DMY 30. Eukaryotic transcriptionElongation and termination Basic mechanism of elongation is essentially the same inprokaryotic and eukaryotic cells- Transcription bubble- Ribonucleotides added onto the 3 end of the growing RNA Eukaryotic transcription termination- Each type of RNA pol utilizes a different terminationmechanism- None of them are as well characterized as termination in bacteria - RNA pol II = No clear termination signal- Transcription continues well beyond the end of the gene- However, there are characteristic termination sequences(rich in As and Ts)- Termination is coupled to mRNA processing (see later) 31. Concept Check 3What is the difference between the core promoterand the regulatory promoter? a. Only the core promoter has consensussequences. b. The regulatory promoter is farther upstream fromthe gene. c. Transcription factors bind to the core promoter;transcriptional activator proteins bind to theregulatory promoters. d. Both b and c above 32. Concept Check 3What is the difference between the core promoterand the regulatory promoter? a. Only the core promoter has consensussequences. b. The regulatory promoter is farther upstream fromthe gene. c. Transcription factors bind to the core promoter;transcriptional activator proteins bind to theregulatory promoters. d. Both b and c above 33. Benjamin A. Pierce GENETICS A Conceptual Approach FOURTH EDITION CHAPTER 14 RNA Molecules and RNA Processing 2012 W. H. Freeman and Company 34. Chapter 14 Outline14.1 Many Genes Have Complex Structures, 37614.2 Messenger RNAs, Which Encode the AminoAcid Sequences of Proteins, Are Modified afterTranscription in Eukaryotes, 37914.3 Transfer RNAs, Which Attach to Amino Acids,Are Modified after Transcription in Bacteria andEukaryotic Cells, 389 35. Gene Organization The concept of colinearity and noncolinearity 36. The Concept of the Gene The gene includes DNA sequence that codes forall exons, introns, and those sequences at thebeginning and end of the RNA that are nottranslated into a protein, including the entiretranscription unit the promoter, the RNAcoding sequence, and the terminator. 37. The structure of messenger RNA A mature mRNA contains 5 untranslated region (5UTR, or leader sequence) ShineDalgarno sequence (ribosomal binding site) Protein coding region 3 untranslated region 38. Regulation of transcription Prokaryotic cells Transcriptional regulation in prokaryotic cells 1) Promoters and sigma factors- As stated earlier, bacteria contain many different types sigma factors- Different factors recognize different promoters- Some factors allow for high levels of transcription, others onlypromote low levels- When a cell needs to transcribe specific genes, itadds the appropriate factor to the core components Sends it off 39. Eukaryotic and prokaryotic cells have the ability to control how often specificgenes are transcribed (which would ultimately control protein levels)- Example: Dont need/want liver specific genes transcribed in brain cells}Liver specificgenes{ 40. mRNA processing 41. Gene Organization Introns Exons 42. Pre-mRNA processing The Addition of the 5 cap: A nucleotide with 7-methylguanine; 5-5bond is attached to the 5-end of the RNA. The Addition of the poly(A) tail: 50 ~ 250 adenine nucleotides are added tothe 3-end of the mRNA. 43. Eukaryotic mRNA processingCapping of the mRNA Eukaryotic mRNA is modified prior to leaving the nucleus 1) Addition of a cap to the front (5) end- The 5 end of the growing mRNA is modifiedvery soon after the start of transcription - Steps: a) A guanine is added to the absolute 5 endvia a 5-5 linkage to the 1st nucleotide- Different from the normal 5-3 phosphodiester linkagesb) That guanine and the 1st few nucleotides are then methylatedc) Cap-binding proteins then attach to the cap - Cap-binding proteins function to:1) Protect the mRNA from RNases in the cytoplasm2) Indirectly allow mRNA to attach to the small ribosomal subunit 44. mRNA processingA Addition of the poly A tailAAAAAAA Eukaryotic mRNA is modified prior to leaving the nucleus 2) Addition of a poly-A tail to the back (3) end- Most genes are transcribed beyond the coding sequence (sometimes>1,000 extra) - The extra sequence will be cut off and a poly A tail will be added - Steps:a) An enzyme (poly(A) polymerase) detects a consensus sequence AAUAAA near the end of mRNA and cuts it ~25 nucleotides downstreamb) The enzyme then adds 50-200 adenines to the cut end - The string of adenines (poly A tail) function to:1) Protect the 3 end of the mRNA from RNases2) Allows cell to regulate mRNA stability - Longer the poly A tail Longer life span3) Help in mRNA-ribosome binding 45. Pre-mRNA processing Nuclear organization Intron removal, mRNA processing, and transcription take place at the same site in the nucleus. Minor Splicing Self-splicing introns happen in some rRNA genes inprotists and in mitochondria genes in fungi. Alternative processing pathways for processing pre-mRNA. 46. mRNA processing Removal of introns Eukaryotic mRNA is modified prior to leaving the nucleus3) Cutting out of INTRONS from the mRNA- Most eukaryotic genes contain stretchesof noncoding sequence (introns) betweencoding sequences (exons) - Most genes split into good and useless parts- Where did they come from?- Retroviruses/transposons or mutated portionof a former exon (lose part of the gene only) - Some properties of introns: a) Common in eukaryotes, rare in prokaryotes b) More complex the organism, more complex/abundant the introns c) Intron abundance and size vary per gene within a species - Some genes have no introns, others have as many as 60 THEY MUST BE REMOVED FROM THE mRNA!!! 47. mRNA processingRemoval of introns Eukaryotic mRNA is modified prior to leaving the nucleus 3) Cutting out of INTRONS from the mRNA- Introns are classified by how they are removed- Some types of introns:a) Group I and II introns- Found in rRNA genes and a few bacterial genes(generally small introns)- Uses a molecule of guanosine to excise itself out and glue the remaining exons together b) Nuclear pre-mRNA introns- Larger and more complex than Group I/II- Require help from an enzyme complex calledthe splicesome in order to remove its introns - Usually contain consensus sequences at the borders attract the splicesome 48. mRNA processing Removal of introns Eukaryotic mRNA is modified prior to leaving the nucleus 3) Cutting out of INTRONS from the mRNA- Alternative splicing of introns/exons can yield different proteins- Some mRNAs can be spliced in different ways - All introns removed, exons are differentially cut out/retained- Different mRNAs Different protein products (often called isoforms) - The more exons, the more potential different isoforms- Example- Tropomyosin gene has 14 exons 10 different protein isoformsONE GENEONE PROTEIN opposite of what I told you previously 49. Concept Check 2Alternative 3 cleavage sites result in:a. multiple genes of different length.b. multiple genes of pre-mRNA of different length.c. multiple mRNAs of different length.d. all of the above. 50. Concept Check 2Alternative 3 cleavage sites result in:a. multiple genes of different length.b. multiple genes of pre-mRNA of different length.c. multiple mRNAs of different length.d. all of the above. 51. Nuclear export of processed mRNA Processed eukaryotic RNAs will only exit the nucleus witha cap, poly A tail, and no introns Exit through nuclear pore complexes - Specific proteins called mRNPs associate with processed mRNA molecules and direct them to and through the nuclear pore - mRNPs interact with a pore complexes called the mRNA exporter- Other proteins are involved mRNAs that fail to be spliced (introns removed) willnot exit the nucleus- Why is this important? Protein will not be made 52. Transcription - Recapitulation All genes (eukaryotic and prokaryotic) have a unique region (promoters) of DNA sequenceupstream of (before) the transcriptional start site that serves as a binding site for the RNApolymerase enzyme General functions of promoters:1. Provides specificity Each gene has a unique promoter sequence2. Tell RNA polymerase where to start (they are usually at the beginning)- No promoter (or mutated promoter), no transcription3. Indicate which strand will be transcribed and the direction of transcription Most promoters share a few common sequences called consensus sequencesProkaryotic promoters share 2 main types of consensus sequences- TATA box (aka Pribnow box) (-10-15), sequence is usually TATAAT- TTGACA found 35 nucleotides upstream RNA polymerase enzyme makes direct contact with the promoter at these consensussequences Unique seq. within and around promoters help determine rates of transcriptionProkaryotic cells only produce 1 type of RNA polymerase to produce all of the mRNA, tRNA,and rRNA in the cell. Prokaryotic RNA polymerase structure:- 2 alpha (), 1 beta (), 1 beta prime (), and 1 sigma () subunit- Together they have the basic catalytic function of producing new RNA- factor controls binding to the promoter (PROMOTER RECOGNITION) - After initial binding has been successful, the factor comes off the core - Bacteria make many different types of sigma factors - Different sigma factors recognize different promoter sequences. 53. Transcription - Recapitulation Prokaryotic RNA polymerase structure:- , , subunits interact and form the CORE ENZYME- Together they have the basic catalytic function of producing new RNA - factor controls binding to the promoter (PROMOTER RECOGNITION)- After initial binding has been successful, the factor comes off the core- Bacteria make many different types of sigma factors- Different sigma factors recognize different promoter sequences Once it binds to the specific promoter, RNA polymerase positions itself over the transcriptional start site based on its distance from the consensus sequences - No specific start signal exists RNA pol unwinds the DNA near the start site (transcription bubble created). RNA pol continues producing a complimentary RNA molecule until it transcribesa terminator signal.Two major prokaryotic transcriptional terminator signals exist - Both must: (a) Slow down the RNA polymerase; (b) Weaken the interaction between the DNAand RNA in the bubble.Properties of the two major prokaryotic terminator signals1. Rho-independent terminators - The DNA sequence contains an inverted repeat followedby a string of 6 adenines. Following their transcription, the inverted repeats form Hydrogenbonds with each other within the RNA and form a hairpin loop - Loop formation causes the RNA pol to pause- The pause combined with weak hydrogen bonding between 6 straight A-U pairs cause the RNA to totally fall off of the DNA template 54. Transcription - Recapitulation ( two major prokaryotic terminator signals)2. Rho-dependent terminators - Also contain inverted repeats that cause hairpinloop formation in the new RNA. Slows down the polymerase - RNA contains a binding site for the Rho protein. Rho moves towards the 3 end - Acts as a RNA/DNA helicase. Rho is ATPase and helicase. - Many prokaryotic genes are organized in operons (genes in tandem, in similar pathway).Eukaryotic transcription follows the same basic steps of prokaryotic transcription- Activation, initiation, elongation, termination- Major differences exist in how they accomplish the above steps General features of transcriptional activation is similar in prokaryotic and eukaryotic cells - Some genes are constituitivelly expressed, others are regulated - The exact nature of the signals that activate transcription can be very different Initiation Making the DNA accessible. Eukaryotic DNA is tightly coiled around proteinscalled histones (and some nonhistone proteins)- DNA is negatively charged (phosphate groups), histones are very positively charged- A histone octamer (8 histone proteins in a complex). Each group is called a nucleosome DNA is freed from histones in 2 major ways:1) Histone acetylation (HATs) add an acetyl group (CH3CO) to histones. This neutralizes theirpositive charge and causes them to lose their attraction for DNA.- Occurs only in the area to be transcribed (it is specfic!) Deacetylases remove acetyl groups after transcription 2) Chromatin remodeling - Some proteins move nucleosomes around without directlymodifying histones.