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Textbook Notes for Lecture 5 and 6

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John Coleman

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Transcription elongation in eucaryotes is tightly couples to RNA processing: In eucaryotes, transcription’s only the first of several steps needed to produce an mRNA; other steps are covalent modifications of RNA ends and removal of intron sequences that are discarded from the middle of the RNA transcript by the process of RNA splicing - Both eukaryotic ends are modified by capping on the 5’ end and by polyadenylation of the 3’ end these special ends allow cell to evaluate whether both ends of an mRNA molecule are present (so message’s intact) before it sends the RNA sequence from the nucleus and translates it into protein - RNA splicing joins the different portions of protein coding sequence and provides higher eucaryotes with ability to synthesize many different proteins from the same gene • Phosphorylation of RNA polymerase II tail (C-terminal domain) is key in transcription initiation by RNA polymerase II ↓ This phosphorylation proceeds gradually as RNA polymerase initiates transcription and moves along DNA  this helps to dissociate the RNA polymerase II from other proteins present at the transcription start point; and allows a new set of proteins to associate with the RNA polymerase tail that function in transcription elongation and RNA processing ↓ Somw of these processing proteins “hop” from the polymerase tail onto the nascent RNA molecule to begin processing it as it emerges from the RNA polymerase The CTD is almost 10 times longer than the remainder of the RNA polymerase so it serves as a tether holding proteins close by until they’re needed this speeds up the rate of further reactions RNA capping is the first modification of eukaryotic pre-mRNAs: - as soon as RNA polymerase II has produced 25 nucleotides of RNA, the 5’ end of the new RNA molecule is modified by addition of a cap that has a modified guanine nucleotide - 3 enzymes that act in succession, perform the capping: one (a phosphate) removes a phosphate from the 5’ end of the nascent RNA, another (guanyl tranferase) adds a GMP in a reverse linkage (5’ to 5’ instead of 5’ to 3’) and a 3 (methyl transferase) adds a methyl group to guanosine ↓ All 3 enzymes bind to RNA polyemerase tail phosphyralted at serine-5 position and so the modification added by TFIIH during transcription initiation, they are ready to modify the 5’ end of the nascent transcript as soon as it emerges from the polymerase • the 5’-methyl cap signifies the 5’ end of eukaryotic mRNAs and this helps the cell to distinguish mRNAs from the other types of RNA molecules in cell ex. RNA polymerase I and III produce uncapped RNAs during transcription because they lack CTD. In nucleus cap binds a protein complex called CBC (cap-binding complex) which helps RNA to be properly processed and exported RNA splicing removes intron sequences from newly transcribed pre-mRNAs: Protein coding sequences of eukaryotic genes are usually interrupted by noncoding intervening sequences (introns) - eukaryotic genes are broken up into small pieces of coding sequence (exons) scattered with much longer intervening sequences (introns); thus the coding portion of eukaryotic gene is often only a small part of the length of the gene - both intron and exon sequences are transcribed into RNA a)A small beta-globin gene, which encodes one of the subunits of the oxygen-carrying protein haemoglobin contains 3 exons. B) the larger Factor VII gene contains 26 exons which codes for a protein - intron sequences are removed from newly synthesized RNA through RNA splicing - only after 5’ and 3’ end processing and splicing have happened then the RNA is an mRNA  each splicing event removes one intron proceeding through 2 sequential phosphoryl-transfer reactions known as transesterifications joins 2 exons while removing the introns as a “lariat” ↓ These reactions could take place without nucleoside triphosphate hydrolysis because the number of high-energy phosphate bonds remains the same The machinery that catalyzes the pre-mRNA splicing has 5 additional RNA molecules and as many as 200 proteins and hydrolyzes many ATP molecules per splicing event this ensures that splicing’s accurate α- Tropomyosin is a coiled-coil protein that regulates contraction in muscle cells. Primary transcript can be spliced in different ways to produce different mRNAs which then give rise to variant proteins. Some of the splicing patterns are specific for certain types of cells. The arrows - presence of many introns in DNA in top part mark the site where cleavage and poly-A additioallows the 3’ genetic recombination to readily ends of the mature mRNAs. combine the exons of different genes which allows genes for new proteins to evolve more easily by the combination of parts of pre-existing genes genes - transcripts of many eukaryotic genes are spliced in more than one way so it allows the same gene to produce a corresponding set of different proteins so, RNA splicing allows eukaryotes to increase the coding potential of their genomes Nucleotide sequences signal where splicing occurs: Mechanism of pre-mRNA splicing implies that splicing machinery must recognize 3 portions of the precursor RNA molecule: the 5’ splice site, the 3’ splice site, and the branch point in the intron sequence that forms the base of the excised lariat - each site has a consensus nucleotide sequences that’s similar from intron to intron and provides the cell with cues for where the splicing is to take place these consensus sequences are short and can accommodate a high degree of sequence variability; the cell incorporates additional types of information to ultimately choose exactly where on each RNA molecule splicing is to take place - the possibility of alternative splicing gives the problem of predicting protein sequences solely from a genome sequence ↓ This is one of the main barriers to identifying all of the genes in a complete genome sequence and of the main reasons why we know only an estimate of the number of genes in the human genome RNA-processing enzymes generate the 3’ end of eukaryotic mRNAs: - the 5’ end of the pre-mRNA produced by RNA polymerase II is capped as soon as it emerges from the RNA polymerase ↓ Then, as polymerase continues its movement along a gene, the spliceosome assembles on the RNA and delineates the intron and exon boundaries - the long C-terminal tail of RNA polymerase coordinates these processes by transferring capping and splicing components directly to the RNA as it emerges from the enzyme ↓ As RNA polymerase II reaches the end of a gene, a similar mechanism ensures that the 3’ end of the pre-mRNA is appropriately processed - the position of the 3’ end of each mRNA molecule is specified by a signal encoded in the genome  these signals are transcribed into RNA as the RNA polymerase II moves through them and they are then recognized (as RNA) by a series of RNA-binding proteins and RNA-processing enzymes - 2 multisubunit proteins called CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor) are important they travel with the RNA polymerase tail and are transferred to the 3’ end processing sequence on an RNA molecule as it emerges from the RNA polymerase - Once CstF and CPSF bind to specific nucleotide sequecnes on the emerging RNA molecule, additional proteins assemble with them to create the 3’ end of the mRNA 1. RNA is cleaved 2. An enzyme called poly-A polymerase (PAP) adds, one at a time, almost 200 A nucleotides to the 3’ end produced by the cleavage - The nucleotide precursor for these additions is ATP and the same type of 5’-3’ bondsa re formed as in conventional RNA synthesis - PAP doesn’t need a template so the poly-A tail of eukaryotic mRNAs is not directly encoded in the genome 3. As poly-A tail’s synthesized proteins called poly-A binding proteins assemble onto it and determine the final length of the tail 4. After 3’end of eukaryotic pre-mRNA molecule cleaved RNA polymerase II continues to transcribe ↓ Polymerase soon releases its grip on the template and transcription terminates 5. The newly synthesized RNA that emerges from the polymerases lack a 5’ cap; this unprotected RNA’s rapidly degraded by a 5’3’ exonuclease which is carried along on the polymerase tail  This RNA degradation causes the RNA polymerase to dissociate from DNA Mature eukaryotic mRNAs are Generating the 3’ end of a eukaryotiselectively exported from the nucleus: mRNA - when the introns of an organism is longer than the exons, a problem is created - of the pre-mRNA synthesized, only a small fraction (the mature mRNA) is of use for cell, the rest—excised introns, broken RNAs and aberrantly processed pre-mRNAs is useless and dangerous Distinguishing between rare mature mRNA molecules it wants to keep and the large debris from RNA processing: - As RNA molecule’s processed, it loses certain proteins and acquires others, thus signifying the successful completion of each of the different steps - A properly completed mRNA molecule is also distinguished by the proteins it lacks Ex. The presence of a snRNP would signify incomplete or aberrant splicing only when the proteins present on an mRNA molecule collectively signify that processing was successfully completed is the mRNA exported from the nucleus into the cytosol, where it can be translated into protein ↓ Improperly processed mRNAs and other RNA debris are retained in the nucleus where they are eventually degraded by the nuclear exosome, a large protein complex whose interior is rich in 3’-to-5’ RNA exonuclease ↓ Eukaryotic cells thus export only useful RNA molecules to the cytoplasm while debri’s trashed in the nucleus • The most abundant of the proteins that assemble on pre-mRNA molecules as they emerge from transcribing RNA polymerases are hnRNPs some of these proteins unwind the hairpin helices from the RNA so that splicing and other signals on the RNA can be read more easily. Others package the RNA contained in the very long intron sequences usually found in genes of complex organisms— they might play a key role in distinguishing mature mRNA from the debris left over from RNA processing • Successfully processed mRNAs are guided through the nuclear pore complexes (NPCs) which are aqueous channels in nuclear membrane that directly connect the nucleoplasm and cytosol  Small molecules can diffuse freely through these channels but most of the macromolecules in cells including mRNAs complexed with proteins are far too large to pass through the channels without a special process - The cell uses energy to actively transport such macromolecules in both directions through the nuclear pore complexes Macromolecules moved through nuclear pore complexes by nuclear transport receptors which depending on the identity of the macromolecule, escort it from the nucleus to the cytoplasm or vise versa - In order for mRNA export to occur a specific nuclear transport receptor must be loaded onto the mRNA, a step that takes place in concert with 3’ cleavage and polyadenylation ↓ Once it helps to move an RNA molecule through the nuclear pore complex, the transport receptor dissociates from the mRNA, re-enters the nucleus and exports a new mRNA molecule Export-ready mRNA molecule and its transport through the nuclear pore Some proteins travel with mRNA as it moves through the pore, whereas others remain in the nucleus. The nuclear export receptor for mRNAs is a complex of proteins that’s deposited when the mRNA’s been correctly spliced and polyadenylated. When mRNA’s exported to cytosol, export receptor dissociates from the mRNA and is re-imported into the nucleus, where it can be used again. After it leaves the nucleus and before it loses the cap-binding complex (CBC) the mRNA is subjected to a final check, called nonsense-mediated decay. Once it passes this test, the mRNA continues to shed previously The definition of a gene has had to be modified since the discovery of alternative RNA splicing: - Gene was defined in molecular terms from work on biochemical genetics of fungus neurospora  until then, a gene had been defined operationally as a region of the genome that segregates as a single unit during meiosis and gives rise to a definable phenotypic trait, such as red or white eye in Drosophilia - Work on neurospora showed that most genes correspond to a region of the genome that directs the synthesis of a single enzyme this led to hypothesis that one gene encodes one polypeptide chain gene becaome identified as that stretch of DNA that was transcribed into the RNA coding for a single polypeptide chain (or a single structural RNA such as tRNA or an rRNA molecule). - It’s now clear that many DNA sequences in higher eukaryotic cells can produce a set of distinct but related proteins by means of alternative RNA splicing - In rare cases where a single transcription unit creates 2 very different eukaryotic proteins, the 2 proteins are considered to be produced by distinct genes that overlap on the chromosome ↓ So, a better way is to modify the original definition to count as a gene any DNA sequence that’s transcribed as a single unit and encodes one set of closely related polypeptide chains (protein isoforms)  this definition also accommodates those DNA sequences that encode protein variants produced by post-transcriptional processes other than RNA splicing, such as translational frame shifting, regulated poly-A addition, and RNA editing Sex determination in Drosophilia depends on a regulated series of RNA splicing events: In drodophilia, the primary signal for determining whether the fly develops as a male or female is the ratio of the # of X chromosomes to the # of autosomal sets (A) - Individuals with an X/A ratio of 1 (ie. 2 X chromosomes and 2 sets of autosomes) develops as a female, and those with an X/A ratio of 0.5 (ie. One chromosome over 2 sets of autosomes) develops as a male ↓ This ratio’s assessed early in development and is remembered thereafter by each cell - 3 important gene products transmit info about this ratio to the many other genes that specify male and female characteristics  Sex determination in Drosophilia depends on a flow of regulated RNA splicing events that involves these 3 gene products  Other organisms use an entirely different scheme for sex determination—one that’s based on transcriptional and translational controls  Drosophilia male-determination pathway needs a number of non-functional RNA molecules to be continually produced which is wasteful one guess for this is that this RNA-splicing cascade like  riboswitches, represents an ancient control strategy left over from the early stage of evolution in which RNA was the predominant biological molecule and controls of gene expression would have had to be based almost entirely on RNA-RNA interactions The cascade of changes in gene expression that determines sex of fly through alternative RNA splicing X chromosome/autosome set ratio of 0.5 results in male development. Male is default pathway where Sxl and Tra genes are both transcribed but the RNAs are spliced constitutively to produce only non-functional RNA molecules and the Dsx transcript is spliced to produce a protein that turns off the genes that specify female characteristics. X/A ratio of 1 triggers female differentiation pathway in embryo by transiently activating promoter within Sxl gene that causes synthesis of a special class of Sxl transcripts that are constitutively spliced to give functional Sxl protein. Sxl is a splicing regulatory protein with 2 sites of action: 1) it binds to the constitutively produced Sxl RNA transcript causing a RNA transport from the nucleus can be regulated: female-specific splice that continues the production of a functional Sxl protein; 2) it binds to the constitutively produced Tra RNA and causes an alternative splice of this transcript which now produces an active Tra - In mammalian cells, about 1/20 of total mass of regulatory protein. Tra protein acts with the constitutively produced Tra2 RNA synthesized ever leaves the nucleus protein to produce the female-specific spliced form of the Dsx transcript; this encodes the female form of Dsx protein, which turns off genes that - Incompletely processed and otherwise specify male features. damaged RNAs are also eventually degraded as part of the quality control system of RNA production - Export of RNA molecules from nucleus is delayed until processing has been completed - Mechanisms that override this control point can be used to regulate gene expression this forms the regulated nuclear transport of mRNA which occurs in the human AIDS virus, HIV - Once inside the cell, HIV directs the formation of a double-stranded DNA copy of its genome, which is then inserted into the genome of the host ↓ Inside, viral DNA is transcribed as one long RNA molecule by the host cell’s RNA polymerase II ↓ This transcript’s spliced in many way to produce 30 different species of mRNA which is translated into a variety of different proteins - In order to make progeny virus, entire, unspliced viral transcripts must be exported from the nucleus to the cytosol wher they’re packaged into viral capsids and searve as viral genome ↓ This large transcript as well as alternatively spliced HIV mRNAs that the virus needs to move to the cytoplasm for protein synthesis, still carries complete introns host cell’s normal block to nuclear export of unspliced RNAs therefore present a problem to HIV ↓ This block’s overcome by: - Virus encodes a protein (Rev protein) that binds to a specific RNA sequence (Rev responsive element RRE) located within a viral intron Rev protein interacts with a nuclear export receptor (exportin 1) which directs the movement of viral RNAs through the nuclear pores into the cytosol even with introns are present Regulation of nuclear export by the HIV Rev protein Early in HIV infection: (A) only the fully spliced RNAs which contain the coding sequences for Rev, Tat, and Nef are exported from the nucleus and translated. Once sufficient Rev protein has accumulated and been transported into the nucleus (B) unspliced viral RNAs can be exported from the nucleus. Many of these RNAs are translated into proteins and the full-length transcripts are packaged into new viral particles. Regulation of nuclear export by Rev has many important consequences for HIV growth and pathogenesis: - Ensures nuclear export of specific unspliced RNAs, it divides the viral infection into an early plase ( in which Rev is translated from a fully spliced RNA and all of intron-containging viral RNAs are retained in the nucleus and degraded) and a late phase ( unspliced RNAs are exported due to Rev function) ↓ This timing helps virus replicate by providing the gene products in roughly the order in which they’re needed - Helps HIV virus to achieve latency, a
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