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CH 13 Transcription

Indiana University
Uploaded: 3 years ago
Contributor: josryeol
Category: Genetics
Type: Lecture Notes
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Filename:   CH 13 Transcription.pptx (4.58 MB)
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CH 13
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RNA, Transcription and Processing Lecture 13 Outline RNA Structure & Assembly Types of RNAs Gene structure RNA Polymerase Transcription Four Basic Steps Prokaryotic v Eukaryotic Differences Enhancers and Silencers Post-Transcriptional Message Processing RNA Structure RNA ribonucleotides are composed of a sugar, nucleotide base, and one or more phosphate groups, with two critical differences compared to DNA nucleotides The bases adenine, guanine, and cytosine are the same, but thymine is replaced by uracil The sugar ribose is used rather than deoxyribose RNA Assembly and Structure The similar sugars in RNA and DNA lead to formation of nearly identical sugar-phosphate backbones in the molecules RNA strands are assembled by formation of phosphodiester bonds between the 5? phosphate of one nucleotide and the 3? hydroxyl of the adjacent nucleotide RNA is synthesized from a DNA template using complementary base pairing (A with U and C with G) RNA polymerase catalyzes the addition of each ribonucleotide to the 3? end of the nascent strand, and form the phosphodiester bonds between nucleotides Two phosphates are eliminated in the process, as in DNA synthesis Transcription Simple Video https://youtu.be/5MfSYnItYvg RNA Types and Classification Messenger RNA (mRNA) is a short-lived intermediary between DNA and protein Transfer RNAs (tRNAs) are encoded in dozens of forms and are responsible for binding an amino acid and depositing it for inclusion into a growing protein chain Ribosomal RNA (rRNA) combines with numerous proteins to form ribosomes Small nuclear RNA (snRNA) of various types is found in the nucleus of eukaryotes and plays a role in mRNA processing Micro RNA (miRNA) is active in plant and animal cells and is involved in postranscriptional regulation of mRNA Small interfering RNA (siRNA) protects plant and animal cells from production of viruses and movement of transposons Gene Structure The gene contains several segments with distinct functions The promoter is immediately upstream (5?) to the start of transcription, referred to as the ?1 nucleotide The promoter controls the access of RNA polymerase to the gene The coding region of the gene is the portion that contains the information needed to synthesize the protein product The termination region of the gene regulates cessation of transcription The termination region is immediately downstream (3?) to the coding segment of the gene RNA Polymerase Composition The bacterial RNA polymerase holoenzyme is composed of a pentameric core enzyme that binds a sixth subunit, called the sigma (s) subunit The large core enzyme is composed of two a subunits, one b and one b? and an w subunit The core enzyme can transcribe RNA from a DNA template but cannot bind the promoter or initiate RNA synthesis without the s subunit Several different types of sigma subunits, called alternative sigma subunits These alter core enzyme conformation in slightly different ways to facilitate association with different promoter regions in bacteria. helps decide if genes will be on or off like transcription factors Four-Stages of Bacterial Transcription Transcription is the synthesis of a single-stranded RNA molecule by RNA polymerase The polymerase uses the template strand of the DNA to assemble a complementary, antiparallel strand of ribonucleotides The coding strand of DNA, also called the nontemplate strand, is complementary to the template strand Promoter recognition Transcription initiation Chain elongation Chain termination Promoter recognition Consensus sequences are written in single-stranded shorthand form, 5? to 3? on the coding strand At the ?10 position is the Pribnow box, or ?10 consensus sequence, 5?-TATAAT-3? At ?35 is a 6-bp region, the ?35 consensus sequence, 5?- TTGACA-3? RNA polymerase binds to ?10 and ?35 sequences and occupies the space between and around them Transcription Initiation First, the holoenzyme makes a loose attachment to the promoter sequence to form the closed promoter complex The holoenzyme next unwinds about 18 bp of DNA around the ?10 position to form the open promoter complex Next, the holoenzyme progresses downstream to initiate RNA synthesis at the ?1 site Considerable sequence variation exists among promoters; alternative sigma sites allow for holoenzyme binding to variant promoters Transcription Elongation The holoenzyme initiated RNA synthesis at the ?1 site It remains intact until the first 8 to 10 RNA nucleotides have been joined, at which point the sigma subunit dissociates from the core enzyme DNA is unwound ahead of the enzyme to maintaining about 18 nucleotides of unwound DNA; the double helix reforms behind the RNA polymerase Termination of Transcription When transcription of the gene is completed, the 5? end of the RNA trails off the core enzyme The core enzyme dissociates from the DNA Shortly after one round of transcription is initiated, a second round begins Termination: Intrinsic Most bacterial termination occurs via intrinsic termination Termination sequences include an inverted repeat followed by a string of adenines mRNA containing the inverted repeats form into a short stem-loop structure, called a hairpin The hairpin followed by a series of Us in the mRNA causes the RNA polymerase to slow down and destabilize The instability caused by the slowing polymerase and the U-A base pairs induces the polymerase to release the transcript and separate from the DNA Termination: Rho-Dependent Certain bacterial genes require the action of rho protein to bind to the nascent mRNA and catalyze the separation of the mRNA from the RNA polymerase Rho-dependent termination sequences do not have a string of uracils; instead they have a rho utilization (or rut) site, a stretch of about 50 nucleotides rich in cytosines Rho proteins contains six identical polypeptides with two functional domains each They are activated by ATP binding to one of the functional domains, facilitating binding to the rut site Rho then moves along the transcript to RNA polymerase and catalyzes the breakage of hydrogen bonds between the mRNA and the DNA template, and release of the polymerase End Product Eukaryotic Transcription Differences Multiple polymerases Eukaryotic promoters and consensus sequences are more diverse than those of bacteria Eukaryotes have three different RNA polymerases that recognize different promoters and produce different types of RNAs The complex that assembles to initiate and elongate transcription is more complex in eukaryotes than in bacteria Eukaryotic genes carry introns and exons, and require processing to remove introns Eukaryote DNA is associated with proteins to form chromatin; the chromatin composition of a gene affects its transcription Chromatin thus plays an important role in gene regulation of eukaryotes Eukaryotic Polymerases RNA polymerase I (RNA pol I) transcribes three ribosomal RNA genes RNA polymerase II (RNA pol II) transcribes protein coding genes and most small nuclear RNA genes RNA polymerase III (RNA pol III) transcribes tRNA, one small nuclear RNA, and one ribosomal RNA Each eukaryotic (and archaeal) RNA polymerase contains units that share homology with the 5 subunits of the bacterial polymerase Arachaea and eukaryotes have 6 to 11 additional subunits All RNA polymerases share a similar “hand” shape with “fingers” that grasp DNA and a “palm” where RNA synthesis takes place Promoter Elements The most common eukaryotic promoter consensus sequence is the TATA box, located at about position ?25 The consensus sequence is 5?-TATAAA-3? A CAAT box is often found near the -80 position A GC-rich box (consensus 5?-GGGCGG-3?) is located at ?90, or further upstream Diversity of Promoter Elements Promoter Recognition RNA pol II recognizes and binds to promoter sequences with the aid of proteins called transcription factors (TFs) TFs bind to regulatory sequences and interact directly, or indirectly, with RNA polymerase; those interacting with pol II are called TFII factors The TATA box is the principle binding site during promoter recognition At the TATA box, TFIID, a multisubunit protein containing the TATA-binding protein (TBP), binds the TATA box sequence and a protein called the TBP-associated factor (TAF) The assembled TFIID bound to the TATA box forms the initial committed complex Next, TFIIB, TFIIF, and RNA pol II join the complex to form the minimal initiation complex The minimal initiation complex is joined by TFIIE and TFIIH to form the complete initiation complex The complete initiation complex contains multiple proteins commonly referred to as “general transcription factors” The complete complex directs RNA pol II to the ?1 position, where it begins to assemble mRNA Detecting Promoter Consensus Elements Research to verify that a segment of DNA is a functionally important component of a promoter has two components Discovering the presence and location of DNA sequences that transcription factors will bind to Mutational analysis to confirm the functionality of each sequence Mutational Analysis of Promoters Researchers produce many different point mutations and compare the level of transcription generated by the mutant sequence relative to wild type Mutations inside the consensus region significantly reduce levels of transcription Mutations outside the consensus region have nonsignificant effects on transcription Enhancers and Silencers Promoters alone may not be sufficient to initiate eukaryotic transcription Two categories of DNA regulatory sequences lead to differential expression of genes These are enhancer sequences and silencer sequences Enhancer Sequences Enhancer sequences increase the level of transcription of specific genes They bind proteins that interact with the proteins that are bound to gene promoters, and together the promoters and enhancers drive gene expression Enhancers may be variable distances from the genes they affect and may be upstream or downstream of the gene Enhancer Sequences and DNA Bending Enhancer sequences bind activator proteins and associated coactivators that form a “protein bridge” that links the proteins at the enhancer sequence to the initiation complex at the promoter This bridge bends the DNA so that the proteins at both locations are brought close enough together for them to interact Silencer Sequences Silencer sequences are DNA elements that act at a distance to repress transcription of their target genes Silencers bind transcription factors called repressor proteins that induce bends in DNA These bends reduce transcription of the target gene Silencers may be located variable distances from their target genes, either upstream or downstream Signal Transduction Signal transduction pathways are sequential events that release regulatory molecules inside a cell in response to events outside the cell They utilize transmembrane proteins, which receive signals externally through an extracellular interaction domain They transmit signals within the cell via a binding domain inside the cell; this activates a transcription factor needed for expression of a target gene RNA Polymerase I Promoters RNA polymerase I transcribes genes for rRNA using a mechanism similar to that of RNA pol II RNA pol I is recruited to upsteam promoter elements following binding of transcription factors, and transcribes ribosomal genes found in the nucleolus The nucleolus is a nuclear organelle containing rRNA and multiple copies of genes encoding rRNA Promoters recognized by RNA pol I have two functional sequences near the start of transcription The core element stretches from ?45 to ?20, and is needed for initiation of transcription; it is bound by sigma-like factor 1 (SL1) protein The upstream control element spans from ?100 to ?150, and increases the level of transcription; it is bound by upstream binding factor 1 (UBF1) Termination in RNA Pol I or Pol III Transcription Each eukaryotic RNA polymerase has a different termination mechanism RNA pol III transcription is terminated similarly to E. coli intrinsic transcription termination It transcribes a terminator sequence that creates a string of uracils in the transcript, though no stem-loop structure forms near it RNA pol I is terminated at a 17-bp consensus sequence that binds transcription-terminating factor 1 (TTF1) A large rRNA precursor transcript is cleaved about 18 nucleotides ahead of the consensus sequence, which does not appear in the mature transcript Post-Transcriptional Processing Modifies RNA Molecules Eukaryotic transcripts are more stable than bacterial transcripts In eukaryotes, transcription and translation are separated in time and location Eukaryotic transcripts have introns, which are not found in bacterial transcripts These features are all related to post-transcriptional modification of eukaryotic transcripts Post-Transcriptional Processing The initial eukaryotic gene mRNA is called the pre-mRNA whereas the fully processed mRNA is called the mature mRNA; modifications include 5? capping 3? polyadenylation Intron splicing Capping 5? mRNA After the first 20 to 30 nucleotides of mRNA have been synthesized, a special enzyme, guanylyl transferase, adds a guanine to the 5? end of the pre-mRNA Additional enzyme action methylates the newly added guanine and may also methylate nearby nucleotides of the transcript The addition of the guanine to the mRNA is called 5? capping STEPS of Capping Guanylyl transferase removes the g phosphate of the 5? end of the mRNA, leaving two phosphates on the message The guanine to be added loses two phosphates to become guanine monophosphate Guanylyl transferase joins the guanine monophosphate to the 5? mRNA by a 5? to 5? triphosphate linkage Functions of the 5? Cap Protection of mRNA from rapid degradation Facilitating transport of mRNA out of the nucleus Facilitating subsequent intron splicing Enhancing translation efficiency by orienting the ribosome on the mRNA Polyadenylation of 3? Pre-mRNA Termination of transcription by RNA pol II is not fully understood The 3? end of the pre-mRNA is created by enzyme action that removes a section of the 3? message and replaces it with a string of adenines This is thought to be associated with the subsequent termination of transcription Steps of Polyadenylation Cleavage and polyadenylation specificity factor (CPSF) binds near the polyadenylation signal sequence—5?-AAUAAA-3?—which is downstream of the stop codon The pre-mRNA is cleaved 15 to 30 nucleotides downstream of the polyadenylation signal sequence The cleavage releases a fragment of the mRNA which is bound by CFI, CFII and CstF; this fragment is later degraded The 3? end of the cut pre-mRNA undergoes enzymatic addition of 20 to 200 adenines through the action of CPSF and PAP After addition of the first 10 adenines, molecules of poly-A-binding protein (PABII) join the adenine tail and increase the rate of addition of adenines Functions of Polyadenylation Facilitating transport of mature mRNA across the nuclear membrane to the cytoplasm Protecting the mRNA from degradation Enhancing translation by enabling the ribosomal recognition of mRNA Some eukaryotic transcripts (e.g., the histone genes) do not undergo polyadenylation Pre-mRNA Intron Splicing Intron splicing requires great precision to remove intron nucleotides accurately Errors in intron removal would lead to incorrect protein sequences Roberts and Sharp shared the 1993 Nobel Prize for their codiscovery of “split genes,” i.e., the presence of intron and exon sequences Splicing Signal Sequences Specific short sequences define the junctions between introns and exons The 5? splice site is at the 5? intron end and contains a consensus sequence with an invariant GU dinucleotide at the 5?-most end of the intron The 3? splice site at the opposite end of the intron has an 11 nucleotide consensus with a pyrimidine rich region and a nearly invariant AG at the 3?-most end The Branch Site A third consensus region, called the branch site, is 20 to 40 nucleotides upstream of the 3? end of the intron It is pyrimidine-rich and contains an invariant adenine called the branch point adenine near the 3? end of the consensus Mutation analysis shows that all three consensus sequences are required for accurate splicing Splicing Spliceosome Composition The spliceosome is a large complex made of multiple snRNPs (snRNPs U1 through U6) The composition is dynamic, changing through the steps of splicing Spliceosome components are recruited to 5? and 3? splice sites by SR proteins; these bind to sequences in exons called exonic splicing enhancers (ESEs), and ensure accurate splicing Coupling of Pre-mRNA Processing Steps Introns appear to be removed one by one, but not necessarily in order The three steps of pre-mRNA processing are tightly coupled The carboxyl terminal domain (CTD) of RNA polymerase II functions as an assembly platform and regulator of pre-mRNA processing machinery Gene Expression Machines Current models suggest that RNA pol II and an array of pre-mRNA processing proteins function as “gene expression machines” The proteins that carry out capping, intron splicing, and polyadenylation associate with the CTD of pol II All three processes are carried out simultaneously Transcription Advanced Video https://youtu.be/SMtWvDbfHLo Take Home Messages RNA Structure & Assembly (i.e. uracil, ribose) Types of RNAs (i.e. mRNA, tRNA etc) Gene structure (i.e. ORF, promoter) RNA Polymerase (i.e. sigma subunits) Transcription Four Basic Steps (i.e. initiation, termination) Prokaryotic v Eukaryotic Differences Enhancers and Silencers (how they influence expression) Post-Transcriptional Message Processing (capping)

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