RNA Synthesis and Processing 8. 8 RNA Synthesis and Processing Transcription in Bacteria Eukaryotic RNA Polymerases and General Transcription Factors.

RNA Synthesis and Processing 8

8 RNA Synthesis and Processing Transcription in Bacteria Eukaryotic RNA Polymerases and General Transcription Factors Regulation of Transcription in Eukaryotes Chromatin and Epigenetics RNA Processing and Turnover

Introduction Regulation of gene expression allows cells to adapt to environmental changes and is responsible for the distinct activities of differentiated cell types that make up complex organisms.

Introduction Transcription is the first step in gene expression, and the initial level at which gene expression is regulated. In eukaryote cells, RNAs are then modified and processed in various ways.

Transcription in Bacteria Studies of E. coli have provided the model for subsequent investigations of transcription in eukaryotic cells. mRNA was discovered first in E. coli; RNA polymerase was first purified from E. coli.

Transcription in Bacteria RNA polymerase catalyzes polymerization of ribonucleoside 5′- triphosphates (NTPs) as directed by a DNA template, in the 5′ to 3′ direction. Transcription initiates de novo (no primer required) at specific sites—this is a major step at which regulation of transcription occurs.

Transcription in Bacteria Bacterial RNA polymerase has five types of subunits. The σ subunit identifies the correct sites for transcription initiation. Most bacteria have several different σ ’s that direct RNA polymerase to different start sites under different conditions.

Figure 8.1 E. coli RNA polymerase

Transcription in Bacteria Promoter: gene sequence to which RNA polymerase binds to initiate transcription. Promoters are six nucleotides long; located at 10 and 35 base pairs upstream of the transcription start site. Consensus sequences are the bases most frequently found in different promoters.

Figure 8.2 Sequences of E. coli promoters

Transcription in Bacteria Experiments show the functional importance of –10 and –35 regions: Genes with promoters that differ from the consensus sequences are transcribed less efficiently. Mutations in these sequences affect promoter function. The σ subunit binds to both regions.

Transcription in Bacteria Initially, the DNA is not unwound (closed-promoter complex). The polymerase then unwinds 12–14 bases of DNA to form an open- promoter complex, allowing transcription. After addition of about 10 nucleotides, σ is released from the polymerase.

Figure 8.3 Transcription by E. coli RNA polymerase (Part 1)

Figure 8.3 Transcription by E. coli RNA polymerase (Part 2)

Transcription in Bacteria During elongation, polymerase maintains an unwound region of about 15 bp. High-resolution structural analysis shows the β and β ′ subunits form a crab-claw- like structure that grips the DNA template. A channel between these subunits contains the polymerase active site.

Figure 8.4 Structure of bacterial RNA polymerase

Transcription in Bacteria RNA synthesis continues until the polymerase encounters a stop signal. The most common stop signal is a symmetrical inverted repeat of a GC- rich sequence followed by seven A residues.

Transcription in Bacteria Transcription of the GC-rich inverted repeat results in a segment of RNA that forms a stable stem-loop structure. This disrupts its association with the DNA template and terminates transcription.

Figure 8.5 Transcription termination

Transcription in Bacteria Alternatively, transcription of some genes is terminated by a specific termination protein (Rho), which binds extended segments of single-stranded RNA.

Transcription in Bacteria Most transcriptional regulation in bacteria operates at initiation. Studies of gene regulation in the 1950s used enzymes involved in lactose metabolism. The enzymes are only expressed when lactose is present.

Transcription in Bacteria Three enzymes are involved: β -galactosidase cleaves lactose into glucose and galactose. Lactose permease transports lactose into the cell. Transacetylase inactivates toxic thiogalactosides that are transported into the cell along with lactose.

Figure 8.6 Metabolism of lactose

Transcription in Bacteria Genes encoding these enzymes are expressed as a single unit, an operon. Two loci control transcription: o (operator), adjacent to transcription initiation site i (outside the operon), encodes a protein that binds to the operator

Figure 8.7 Negative control of the lac operon

Transcription in Bacteria Mutants that don’t produce i gene product express the operon even when lactose is not available. This implies that the normal i gene product is a repressor, which blocks transcription when bound to o. When lactose is present in normal cells, it binds to the repressor, preventing it from binding to the operator, and the genes are expressed.

Transcription in Bacteria The lactose operon illustrates the central principle of gene regulation: Control of transcription is mediated by the interaction of regulatory proteins with specific DNA sequences.

Transcription in Bacteria Cis-acting control elements affect expression of linked genes on the same DNA molecule (e.g., the operator). Other proteins can affect expression of genes on other chromosomes (e.g., the repressor). The lac operon is an example of negative control—binding of the repressor blocks transcription.

Transcription in Bacteria Negative control: the regulatory protein (the repressor) blocks transcription. Positive control: regulatory proteins activate transcription.

Transcription in Bacteria Example of positive control in E. coli: Presence of glucose (preferred energy source) represses expression of the lac operon, even if lactose is also present. This is mediated by a positive control system: If glucose decreases, levels of cAMP increase.

Transcription in Bacteria cAMP binds to the regulatory protein CAP (catabolite activator protein). This stimulates CAP to bind to its target DNA sequence upstream of the lac operon. CAP facilitates binding of RNA polymerase to the promoter.

Figure 8.8 Control of the lac operon by glucose

Eukaryotic RNA Polymerases and General Transcription Factors Transcription in eukaryotes: Eukaryotic cells have three RNA polymerases that transcribe different classes of genes. RNA polymerases must interact with additional proteins to initiate and regulate transcription.

Eukaryotic RNA Polymerases and General Transcription Factors Transcription takes place on chromatin; regulation of chromatin structure is important in regulating gene expression.

Eukaryotic RNA Polymerases and General Transcription Factors Eukaryotic RNA polymerases are complex enzymes, with 12 to 17 different subunits. They all have nine conserved subunits, five of which are related to subunits of bacterial RNA polymerase. Yeast RNA polymerase II is strikingly similar to that of bacteria.

Table 8.1 Classes of Genes Transcribed by Eukaryotic RNA Polymerases

Figure 8.9 Structure of yeast RNA polymerase II

Eukaryotic RNA Polymerases and General Transcription Factors RNA polymerase II synthesizes mRNA and has been the focus of most transcription studies. It requires initiation factors that (in contrast to bacterial σ factors) are not associated with the polymerase.

Eukaryotic RNA Polymerases and General Transcription Factors General transcription factors are proteins involved in transcription from polymerase II promoters. About 10% of the genes in the human genome encode transcription factors, emphasizing the importance of these proteins.

Eukaryotic RNA Polymerases and General Transcription Factors Promoters contain several sequence elements surrounding the transcription sites. The TATA box resembles the –10 sequence of bacterial promoters. Others include initiator (Inr) elements, TFIIB recognition elements (BRE), and downstream elements DCE, MTE, and DPE).

Figure 8.10 Formation of a polymerase II preinitiation complex in vitro (Part 1)

Eukaryotic RNA Polymerases and General Transcription Factors Five general transcription factors are required for initiation of transcription in vitro. TFIID is composed of multiple subunits, including TATA-binding protein (TBP) and other subunits (TAFs) that bind to the Inr, DCE, MTE, and DPE sequences.

Eukaryotic RNA Polymerases and General Transcription Factors Several other transcription factors (TFIIB, TFIIF, TFIIE, and TFIIH) bind in association with the RNA polymerase II to form the preinitiation complex.

Eukaryotic RNA Polymerases and General Transcription Factors Within a cell, additional factors are required to initiate transcription. These include Mediator, a protein complex of 20+ subunits; it interacts with both general transcription factors and RNA polymerase.

Figure 8.11 RNA polymerase II/Mediator complexes and transcription initiation

Eukaryotic RNA Polymerases and General Transcription Factors RNA polymerase I transcribes rRNA genes, which are present in tandem repeats. Transcription yields a large 45S pre- rRNA, which is processed to yield the 28S, 18S, and 5.8S rRNAs.

Figure 8.12 Transcription of the ribosomal RNA gene

Eukaryotic RNA Polymerases and General Transcription Factors Promoters of rRNA genes are recognized by two transcription factors that recruit RNA polymerase I to form an initiation complex: UBF (upstream binding factor) SL1 (selectivity factor 1)

Eukaryotic RNA Polymerases and General Transcription Factors Genes for tRNAs, 5S rRNA, and some of the small RNAs are transcribed by polymerase III. They are expressed from three types of promoters: TFIIIA, TFIIIB, and TFIIIC.

Figure 8.13 Transcription of RNA polymerase III genes

Regulation of Transcription in Eukaryotes Eukaryotic DNA is packaged into chromatin, which limits its availability for transcription. Non-coding RNAs and proteins regulate transcription via modifications of chromatin structure.

Regulation of Transcription in Eukaryotes Many cis-acting sequences regulate expression of eukaryotic genes. These regulatory sequences have been identified by gene transfer assays.

Regulation of Transcription in Eukaryotes Gene transfer assays: Regulatory sequences are ligated to reporter genes that encode easily detectable enzymes, such as firefly luciferase. The regulatory sequence directs expression of the reporter gene in cultured cells.

Figure 8.14 Identification of eukaryotic regulatory sequences

Regulation of Transcription in Eukaryotes Two cis-acting promoters (TATA and GC boxes) were identified in studies of the herpes simplex virus gene that encodes thymidine kinase. cis-acting regulatory sequences are usually located upstream of the transcription start site.

Figure 8.15 A eukaryotic promoter

Regulation of Transcription in Eukaryotes Enhancers: regulatory sequences located farther away from the start site. First identified in studies of the promoter of virus SV40. Activity of enhancers does not depend on their distance from, or orientation with respect to the transcription initiation site.

Figure 8.16 The SV40 enhancer

Figure 8.17 Action of enhancers (Part 1)

Regulation of Transcription in Eukaryotes Enhancers, like promoters, bind transcription factors that then regulate RNA polymerase. DNA looping allows a transcription factor bound to a distant enhancer to interact with proteins associated with the RNA polymerase/Mediator complex at the promoter.

Figure 8.18 DNA looping

Regulation of Transcription in Eukaryotes Example: an enhancer controls transcription of immunoglobulin genes in B lymphocytes. Gene transfer experiments show that the enhancer is active in lymphocytes, but not in other cell types. This regulatory sequence is partly responsible for tissue-specific expression of the immunoglobulin genes.

Regulation of Transcription in Eukaryotes Enhancers usually have multiple sequence elements that bind different regulatory proteins that work together to regulate gene expression. The immunoglobulin heavy-chain enhancer has at least nine sequence elements that are protein-binding sites.

Figure 8.19 The immunoglobulin enhancer

Regulation of Transcription in Eukaryotes Enhancers account for 10% or more of human genomic DNA, emphasizing the importance of these elements. Many mutations linked to human diseases affect enhancers rather than protein-coding sequences. In any given cell type, multiple enhancers work together to regulate individual genes.

Regulation of Transcription in Eukaryotes Activity of any given enhancer is specific for the promoter of its target gene. Specificity is maintained partly by insulators, which divide chromosomes into independent domains and prevent enhancers from acting on promoters located in an adjacent domain.

Regulation of Transcription in Eukaryotes Genomes are divided into discrete chromosomal domains: topologically associating domains (TADs). Enhancers and promoters within a TAD interact frequently with each other, but only rarely with elements in other domains. The main protein that binds insulators in vertebrates is CTCF.

Figure 8.20 Chromosomal domains and CTCF

Regulation of Transcription in Eukaryotes Transcription factor binding sites have been identified by various experiments: Electrophoretic-mobility shift assay: Radiolabeled DNA fragments are incubated with a protein, then analyzed by electrophoresis. Migration of a DNA fragment is slowed by a bound protein.

Figure 8.21 Electrophoreticmobility shift assay

Regulation of Transcription in Eukaryotes Binding sites are usually short DNA sequences (6–10 bp) and they are degenerate: The transcription factor will bind to the consensus sequence, but also to sequences that differ from the consensus at one or more positions.

Regulation of Transcription in Eukaryotes Transcription factor binding sites are shown as pictograms, representing the frequency of each base at all positions of known binding sites for a given factor.

Figure 8.22 Representative transcription factor binding sites

Regulation of Transcription in Eukaryotes Chromatin immunoprecipitation: Cells are treated with formaldehyde to cross-link transcription factors to the DNA sequences to which they were bound. Chromatin is extracted and fragmented. Fragments of DNA linked to a transcription factor can then be isolated by immunoprecipitation.

Figure 8.23 Chromatin immunoprecipitation (Part 1)

Figure 8.23 Chromatin immunoprecipitation (Part 2)

Regulation of Transcription in Eukaryotes One of the first transcription factors to be isolated was Sp1, in studies of SV40 virus, by Tjian and colleagues. Sp1 was shown to bind to GC boxes in the SV40 promoter. This established the action of Sp1 and also suggested a method for purification of transcription factors.

Key Experiment, Ch. 8, p. 279 (1)

Regulation of Transcription in Eukaryotes DNA-affinity chromatography: Double-stranded oligonucleotides with repeated GC box sequences are bound to agarose beads in a column. Cell extracts are passed through the column. Sp1 binds to the GC box with high affinity and is retained on the column.

Figure 8.24 Purification of Sp1 by DNA-affinity chromatography

Regulation of Transcription in Eukaryotes Transcriptional activators, like Sp1, bind to regulatory DNA sequences and stimulate transcription. These factors have two independent domains: one binds DNA, the other stimulates transcription by interacting with other proteins, such as Mediator.

Figure 8.25 Structure of transcriptional activators

Regulation of Transcription in Eukaryotes Many transcription factors have now been identified in eukaryotic cells. About 2000 are encoded in the human genome. They contain many distinct types of DNA-binding domains.

Regulation of Transcription in Eukaryotes DNA binding domains: 1. Zinc finger domain: binds zinc ions and folds into loops (“fingers”) that bind DNA. Steroid hormone receptors have zinc fingers; they regulate gene transcription in response to hormones such as estrogen and testosterone.

Figure 8.26 Examples of DNA-binding domains (Part 1)

Regulation of Transcription in Eukaryotes 2. Helix-turn-helix domain: one helix makes most of the contacts with DNA, the others lie across the complex to stabilize the interaction. Homeodomain proteins are important in regulation of gene expression during embryonic development.

Regulation of Transcription in Eukaryotes 3. Leucine zipper and helix-loop-helix proteins: DNA-binding domains are formed by dimerization of two polypeptide chains. Different members of each family can dimerize with one another— combinations can form an expanded array of factors.

Regulation of Transcription in Eukaryotes Activation domains of transcription factors are not as well characterized. They stimulate transcription by two mechanisms: Interact with Mediator proteins and general transcription factors Interact with coactivators to modify chromatin structure.

Figure 8.27 Action of transcriptional activators

Regulation of Transcription in Eukaryotes Gene expression is also regulated by repressors, which inhibit transcription. In some cases, they simply interfere with binding of other transcription factors. Other repressors compete with activators for binding to specific regulatory sequences.

Figure 8.28 Action of eukaryotic repressors (Part 1)

Regulation of Transcription in Eukaryotes Active repressors have domains that inhibit transcription via protein-protein interactions. These include interactions with specific activator proteins, with Mediator proteins or general transcription factors, and with corepressors that act by modifying chromatin structure.

Figure 8.28 Action of eukaryotic repressors (Part 2)

Regulation of Transcription in Eukaryotes Transcription can also be regulated at elongation. Recent studies show that many genes have molecules of RNA polymerase II that have started transcription but are stalled immediately downstream of promoters.

Regulation of Transcription in Eukaryotes The RNA polymerase II is poised to continue in response to appropriate signals. Many genes on which poised polymerases have been found are regulated by extracellular signals or function during development.

Regulation of Transcription in Eukaryotes Following initiation, the polymerase pauses within about 50 nucleotides due to negative regulatory factors, including NELF (negative elongation factor) and DSIF. Continuation depends on another factor: P-TEFb (positive transcription- elongation factor-b).

Figure 8.29 Regulation of transcriptional elongation (Part 1)

Figure 8.29 Regulation of transcriptional elongation (Part 2)

Chromatin and Epigenetics Because eukaryotic DNA is packaged in chromatin, chromatin structure is a critical aspect of gene expression. Histones can be modified several ways—key mechanisms for regulating gene expression. Many histone modifications are stably inherited when cells divide.

Chromatin and Epigenetics The basic unit of chromatin is the nucleosome: 147 bp of DNA wrapped around two molecules each of histones H2A, H2B, H3, and H4. One molecule of histone H1 is bound to the DNA as it enters the nucleosome core particle.

Figure 6.17 Structure of a chromatosome (Part 1) Repeat fig. 6.17 A here

Chromatin and Epigenetics Chromatin limits availability of DNA for transcription, affecting both transcription factor binding and action of RNA polymerase. Actively transcribed genes are in relatively decondensed chromatin, which can be seen in polytene chromosomes of Drosophila.

Figure 8.30 Decondensed chromosome regions in Drosophila

Chromatin and Epigenetics Chromatin can be altered by histone modifications and nucleosome rearrangements.

Chromatin and Epigenetics Histone acetylation: Amino-terminal tail domains of core histones are rich in lysine and can be modified by acetylation. Actetyl groups are added by histone acetyltransferase (HAT) and removed by histone deacetylase (HDAC).

Figure 8.31 Histone acetylation (Part 1)

Chromatin and Epigenetics Acetylation neutralizes the positive charge of lysine, relaxing chromatin structure and increasing availability of the DNA template for transcription. Transcriptional activators and repressors are associated with HAT and HDAC.

Figure 8.31 Histone acetylation (Part 2)

Chromatin and Epigenetics Histones can also be modified by methylation of lysine and arginine, phosphorylation of serine, and addition of small peptides (ubiquitin and SUMO) to lysine. These modifications occur at specific amino acid residues in the histone tails.

Figure 8.32 Patterns of histone modification

Chromatin and Epigenetics Histone modifications affect gene expression by altering chromatin properties, and by providing binding sites for proteins that activate or repress transcription. Methylated H3 lysine-9 and -27 residues are binding sites for proteins that induce chromatin condensation and formation of heterochromatin.

Chromatin and Epigenetics Promoters and enhancers have distinct chromatin features. They are free of nucleosomes, and thus accessible for binding transcription factors. These regions can be digested with with DNase (DNase hypersensitive sites).

Figure 8.33 Chromatin at promoters and enhancers (Part 1)

Chromatin and Epigenetics Flanking nucleosomes have different histone modifications. Example: Promoters have trimethylated H3 lysine-4; enhancers have a monomethylated form of this lysine.

Figure 8.33 Chromatin at promoters and enhancers (Part 2)

Chromatin and Epigenetics Chromatin remodeling factors are protein complexes that alter contacts between DNA and histones. They can reposition nucleosomes, change conformation of nucleosomes, or eject nucleosomes from the DNA.

Figure 8.34 Chromatin remodeling factors

Chromatin and Epigenetics Following initiation of transcription, elongation is facilitated by elongation factors associated with the phosphorylated C-terminal domain of RNA polymerase II. Elongation factors include histone modifying enzymes and chromatin remodeling factors.

Chromatin and Epigenetics Modified histones serve as binding sites for proteins that themselves catalyze histone modifications. Histone modifications can thus regulate one another, leading to the stable patterns of modified chromatin.

Chromatin and Epigenetics This provides a mechanism for epigenetic inheritance —transmission of information that is not in the DNA sequence. Modified histones are transferred to both progeny chromosomes where they direct similar modification of new histones— maintaining characteristic patterns of modification.

Figure 8.35 Epigenetic inheritance of histone modifications (Part 1)

Chromatin and Epigenetics Example: Some regulatory genes are repressed in certain cells in developing Drosophila; the repression is passed on in subsequent divisions. Repression results from methylation of H3 lysine 27 by the Polycomb proteins.

Chromatin and Epigenetics One complex (PRC1) binds to methylated H3 lysine 27. The other complex (PRC2) methylates H3 lysine 27. As a result, methylation of H3 lysine 27 can spread to adjacent nucleosomes and is maintained when cells divide.

Figure 8.36 Polychrome proteins

Chromatin and Epigenetics DNA methylation is another mechanism of epigenetic control of transcription: Methyl groups are added at the 5-carbon position of cytosines that precede guanines (CpG dinucleotides). This methylation is correlated with transcriptional repression.

Figure 8.37 DNA methylation

Chromatin and Epigenetics Methylation is common in transposable elements; it plays a key role in suppressing their movement. DNA methylation is also associated with transcriptional repression of mammalian genes involved in development and differentiation.

Chromatin and Epigenetics DNA methylation is an important mechanism for epigenetic inheritance. Following DNA replication, an enzyme methylates CpG sequences of a daughter strand that is hydrogen- bonded to a methylated parental strand.

Figure 8.38 Maintenance of methylation patterns

Chromatin and Epigenetics Methylation can be reversed by the TET family enzymes, which catalyze oxidation of 5-methylcytosine to 5- formylcytosine and 5-carboxylcytosine. These are excised and replaced by cytosine via DNA repair.

Figure 8.39 Reversal of methylation

Chromatin and Epigenetics DNA methylation plays a role in genomic imprinting: expression of some genes depends on whether they come from the mother or the father. Example: Gene H19 is transcribed only from the maternal copy. It is methylated during development of male, but not female, germ cells.

Figure 8.40 Genomic imprinting

Chromatin and Epigenetics Transcription can also be regulated by noncoding RNA molecules: miRNAs (20–30 nucleotides) act by the RNA interference pathway to inhibit translation or induce degradation of homologous mRNAs.

Chromatin and Epigenetics Long noncoding RNAs (lncRNAs) (>200 nucleotides): Form complexes with proteins that modify chromatin and recruit these complexes to their sites of transcription, thereby regulating expression of neighboring genes.

Chromatin and Epigenetics In many cases, lncRNAs repress their target genes by forming complexes with Polycomb Repressive Complex 2 (PRC2). Includes Xist lncRNA, which mediates X chromosome inactivation in mammals and several lncRNAs involved in imprinting.

Figure 8.41 Action of lncRNAs (Part 1)

Chromatin and Epigenetics lncRNAs can associate with different chromatin-modifying enzymes and function as repressors or activators. Some lncRNAs act in trans by recruiting chromatin-modifying complexes (e.g., PRC2) to distant target genes. Many other lncRNAs regulate gene expression in multiple ways.

Figure 8.41 Action of lncRNAs (Part 2)

RNA Processing and Turnover Bacterial mRNAs are used immediately for protein synthesis while still being transcribed. But they are an exception; most RNAs must be processed in various ways. Regulation of processing provides another level of control of gene expression.

RNA Processing and Turnover Ribosomal RNAs of both prokaryotes and eukaryotes are derived from a single long pre-rRNA molecule. In prokaryotes, this is cleaved to form three rRNAs (16S, 23S, and 5S). Eukaryotes have four rRNAs; 5S rRNA is transcribed from a separate gene.

Figure 8.42 Processing of ribosomal RNAs

RNA Processing and Turnover tRNAs also start as long precursors (pre-tRNAs) in prokaryotes and eukaryotes. Processing of the 5′ end of pre-tRNAs involves cleavage by the enzyme RNase P. RNase P is a ribozyme—an enzyme in which RNA rather than protein catalyzes the reaction.

Figure 8.43 Processing of transfer RNAs (Part 1)

RNA Processing and Turnover Processing of the 3′ end of tRNAs involves addition of a CCA terminus, the site of amino acid attachment. Bases are also modified at specific positions. About 10% of the bases are modified.

RNA Processing and Turnover Some pre-tRNAs have introns that are removed by splicing. tRNA splicing is mediated by conventional proteins such as endonuclease, rather than catalytic RNAs.

RNA Processing and Turnover In eukaryotes, pre-mRNAs are extensively modified before export from the nucleus. Throughout processing, transport, translation, and degradation, mRNA molecules are associated with proteins to form messenger ribonucleoprotein particles (mRNPs).

RNA Processing and Turnover Transcription and processing are coupled. The C-terminal domain (CTD) of RNA polymerase II plays a key role by serving as a binding site for the enzymes involved in mRNA processing.

RNA Processing and Turnover The 5′ end of the transcript is modified by addition of a 7-methylguanosine cap. The 5′ cap stabilizes the RNA, and aligns it on the ribosome during translation.

Figure 8.44 Processing of eukaryotic messenger RNAs (Part 1)

Figure 8.44 Processing of eukaryotic messenger RNAs (Part 2)

RNA Processing and Turnover At the 3′ end, a poly-A tail is added by polyadenylation. Signals for polyadenylation are the hexanucleotide AAUAAA, and a GU- rich downstream element. Poly-A polymerase then adds about 200 adenines to form the poly-A tail.

Figure 8.45 Formation of the 3′ ends of eukaryotic mRNAs

RNA Processing and Turnover Recognition of the polyadenylation signal leads to termination of transcription, cleavage, and polyadenylation of the mRNA. The RNA that has been synthesized downstream of the site of poly-A addition is degraded.

RNA Processing and Turnover Introns (noncoding sequences) are removed from pre-mRNA by splicing. In mammals, most genes contain multiple introns. Splicing has to be highly specific to yield functional mRNAs.

RNA Processing and Turnover In vitro systems were developed to study splicing: A gene with introns is cloned adjacent to a promoter for a bacteriophage RNA polymerase. Transcription of these plasmids produced pre-mRNAs that, when added to nuclear extracts of mammalian cells, were found to be correctly spliced.

Figure 8.46 In vitro splicing

RNA Processing and Turnover Splicing proceeds in two steps: 1. Cleavage at the 5′ splice site (SS) and joining of the 5′ end of the intron to an adenine within the intron (branch point). The intron forms a loop. 2. Cleavage at the 3′ SS and simultaneous ligation of the exons excises the intron loop.

Figure 8.47 Splicing of pre-mRNA

RNA Processing and Turnover Three sequence elements of pre- mRNAs are important: At the 5′ splice site, at the 3′ splice site, and within the intron at the branch point. Pre-mRNAs contain similar consensus sequences at each of these positions.

RNA Processing and Turnover Splicing takes place in large complexes, called spliceosomes, which have five types of small nuclear RNAs (snRNAs)—U1, U2, U4, U5, and U6. The snRNAs are complexed with 6–10 protein molecules to form small nuclear ribonucleoprotein particles (snRNPs).

RNA Processing and Turnover snRNAs were first identified in the late 1960s, but their function was unknown. In 1979, Lerner and Steitz demonstrated that snRNAs were present as RNA- protein complexes called snRNPs, and suggested that they might function in pre-mRNA splicing.

RNA Processing and Turnover First step in spliceosome assembly: binding of U1 snRNP to the 5′ SS. Recognition of 5′ SS involves base pairing between the 5′ SS consensus sequence and a complementary sequence at the 5′ end of U1 snRNA.

Figure 8.48 Assembly of the spliceosome (Part 1)

Figure 8.48 Assembly of the spliceosome (Part 2)

Figure 8.49 Binding of U1 snRNA to the 5′ splice site

RNA Processing and Turnover U2 snRNP then binds to the branch point. The other snRNPs join the complex and act together to form the intron loop, and maintain the association of the 5′ and 3′ exons so they can be ligated. This is followed by excision of the intron.

RNA Processing and Turnover snRNAs recognize consensus sequences at the branch and splice sites, and also catalyze the splicing reaction. Some RNAs can self-splice: they catalyze removal of their own introns in the absence of other protein or RNA factors.

RNA Processing and Turnover Two groups of self-splicing introns: Group I—cleavage at 5′ SS mediated by a guanosine cofactor. Group II—cleavage of 5′ SS results from attack by an adenosine nucleotide in the intron.

Figure 8.50 Self-splicing introns (Part 1)

Figure 8.50 Self-splicing introns (Part 2)

RNA Processing and Turnover Other splicing factors bind to RNA and recruit U1 and U2 snRNPs to the appropriate sites on pre-mRNA. SR splicing factors bind to specific sequences in exons and recruit U1 snRNP to the 5′ SS. U2AF binds to pyrimidine-rich sequences at the 3′ SS and recruits U2 snRNP to the branch point.

Figure 8.51 Role of splicing factors in spliceosome assembly

RNA Processing and Turnover Alternative splicing: Most pre-mRNAs have multiple introns, thus different mRNAs can be produced from the same gene. This is one way of controlling gene expression, and increases the diversity of proteins that can be encoded.

RNA Processing and Turnover Sex determination in Drosophila is an example of tissue-specific alternative splicing. Splicing of transformer mRNA is regulated by the SXL protein, which is only expressed in females. SXL acts as a repressor that blocks splicing factor U2AF.

Figure 8.52 Alternative splicing in Drosophila sex determination

RNA Processing and Turnover The Dscam gene of Drosophila has four sets of exons; one from each set goes into the spliced mRNA in any combination, yielding 38,016 possible mRNAs. Different forms of Dscam provide neurons with an identity code essential in establishing connections between neurons for brain development.

Figure 8.53 Alternative splicing of Dscam

RNA Processing and Turnover RNA editing: processing (other than splicing) that alters the protein-coding sequences of mRNAs. It involves single base modification reactions, such as deamination of cytosine to uridine and adenosine to inosine.

RNA Processing and Turnover Editing mRNA for apolipoprotein B, which transports lipids in the blood, results in two proteins: Apo-B100, produced in the liver by translation of unedited mRNA Apo-B48, produced in the intestine from mRNA in which a C has been changed to a U by deamination

Figure 8.54 Editing of apolipoprotein B mRNA

RNA Processing and Turnover A-to-I editing: deamination of adenosine to inosine. Results in single amino acid changes in ion channels and receptors on the surface of neurons. Mutants lacking the editing enzyme have a variety of neurological defects.

RNA Processing and Turnover Over 90% of pre-mRNA sequences are introns, which are degraded in the nucleus after splicing. Processed mRNAs are protected by capping and polyadenylation, but the unprotected ends of introns are recognized and degraded by enzymes.

RNA Processing and Turnover Defective mRNAs can also be degraded. Nonsense-mediated mRNA decay eliminates mRNAs that lack complete open-reading frames. When ribosomes encounter premature termination codons, translation stops and the defective mRNA is degraded.

RNA Processing and Turnover Ultimately, RNAs are degraded in the cytoplasm. Levels of any RNA are determined by a balance between synthesis and degradation. Rate of degradation can thus control gene expression.

RNA Processing and Turnover rRNAs and tRNAs are very stable in both prokaryotes and eukaryotes. This accounts for the high levels of these RNAs (greater than 90% of all RNA) in cells.

RNA Processing and Turnover Bacterial mRNAs are rapidly degraded; most have half-lives of 2–3 minutes. Rapid turnover allows a cell to respond quickly to changes in its environment, such as nutrient availability.

RNA Processing and Turnover In eukaryotes, mRNA half-lives vary; less than 30 minutes to 20 hours. Short-lived mRNAs code for regulatory proteins, levels of which can vary rapidly in response to environmental stimuli. mRNAs encoding structural proteins or central metabolic enzymes have long half-lives.

RNA Processing and Turnover Degradation of eukaryote mRNAs is initiated by shortening the poly-A tails. Rapidly degraded mRNAs often contain specific AU-rich sequences near the 3′ ends—binding sites for proteins that can either stabilize them or target them for degradation.

Figure 8.55 mRNA degradation

RNA Processing and Turnover These RNA-binding proteins are regulated by extracellular signals, such as growth factors and hormones. Degradation of many mRNAs is regulated by miRNAs, which stimulate degradation as well as inhibit translation.

RNA Synthesis and Processing 8. 8 RNA Synthesis and Processing Transcription in Bacteria Eukaryotic RNA Polymerases and General Transcription Factors.

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RNA Synthesis and Processing 8. 8 RNA Synthesis and Processing Transcription in Bacteria Eukaryotic RNA Polymerases and General Transcription Factors.

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