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RNA Synthesis and Processing 7. 7 RNA Synthesis and Processing Chapter Outline Transcription in Prokaryotes Eukaryotic RNA Polymerases and General Transcription.

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Presentation on theme: "RNA Synthesis and Processing 7. 7 RNA Synthesis and Processing Chapter Outline Transcription in Prokaryotes Eukaryotic RNA Polymerases and General Transcription."— Presentation transcript:

1 RNA Synthesis and Processing 7

2 7 RNA Synthesis and Processing Chapter Outline Transcription in Prokaryotes Eukaryotic RNA Polymerases and General Transcription Factors Regulation of Transcription in Eukaryotes RNA Processing and Turnover

3 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.

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

5 Transcription in Prokaryotes Studies of E. coli have provided the model for subsequent investigations of transcription in eukaryotic cells. mRNA was discovered first in E. coli and RNA polymerase was purified and studied.

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

7 Transcription in Prokaryotes Bacterial RNA polymerase has five types of subunits. The σ subunit is weakly bound and can be separated from the others. It 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.

8 Figure 7.1 E. coli RNA polymerase

9 Transcription in Prokaryotes Promoter: gene sequence to which RNA polymerase binds to initiate transcription. Promoters are 6 nucleotides long and are located at 10 and 35 base pairs upstream of the transcription start site. Consensus sequences are the bases most frequently found in different promoters.

10 Figure 7.2 Sequences of E. coli promoters

11 Transcription in Prokaryotes 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.

12 Transcription in Prokaryotes 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 ten nucleotides, σ is released from the polymerase.

13 Figure 7.3 Transcription by E. coli RNA polymerase

14 Transcription in Prokaryotes During elongation, polymerase maintains an unwound region of about 15 base pairs. 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.

15 Figure 7.4 Structure of bacterial RNA polymerase

16 Transcription in Prokaryotes 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.

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

18 Figure 7.5 Transcription termination

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

20 Transcription in Prokaryotes 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.

21 Transcription in Prokaryotes 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.

22 Figure 7.6 Metabolism of lactose

23 Transcription in Prokaryotes Genes encoding these enzymes are expressed as a single unit, called an operon. Two loci control transcription:  o (operator), adjacent to transcription initiation site  i (not in the operon), encodes a protein that binds to the operator.

24 Figure 7.7 Negative control of the lac operon

25 Transcription in Prokaryotes 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.

26 Transcription in Prokaryotes 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.

27 Transcription in Prokaryotes 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.

28 Transcription in Prokaryotes Negative control: the regulatory protein (the repressor) blocks transcription. Positive control: regulatory proteins activate rather than inhibit transcription.

29 Transcription in Prokaryotes 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.

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

31 Figure 7.8 Positive control of the lac operon by glucose

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

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

34 Eukaryotic RNA Polymerases and General Transcription Factors Eukaryotic RNA polymerases are complex enzymes, consisting of 12 to 17 different subunits. They all have 9 conserved subunits, 5 of which are related to subunits of bacterial RNA polymerase. Yeast RNA polymerase II is strikingly similar to that of bacteria.

35 Table 7.1 Classes of genes transcribed by eukaryotic RNA polymerases

36 Figure 7.9 Structure of yeast RNA polymerase II

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

38 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.

39 Eukaryotic RNA Polymerases and General Transcription Factors Promoters contain several different sequence elements surrounding their 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).

40 Figure 7.10 Formation of a polymerase II preinitiation complex in vitro (Part 1)

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

42 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.

43 Figure 7.10 Formation of a polymerase II preinitiation complex in vitro (Part 2)

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

45 Figure 7.11 RNA polymerase II/Mediator complexes and transcription initiation

46 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.

47 Figure 7.12 The ribosomal RNA gene

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

49 Figure 7.13 Initiation of rDNA transcription

50 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.

51 Figure 7.14 Transcription of RNA polymerase III genes

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

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

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

55 Figure 7.15 Identification of eukaryotic regulatory sequences

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

57 Figure 7.16 A eukaryotic promoter

58 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.

59 Figure 7.17 The SV40 enhancer

60 Figure 7.18 Action of enhancers

61 Regulation of Transcription in Eukaryotes Enhancers, like promoters, function by binding 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.

62 Figure 7.19 DNA looping

63 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.

64 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 distinct sequence elements that serve as protein-binding sites.

65 Figure 7.20 The immunoglobulin enhancer

66 Regulation of Transcription in Eukaryotes The immunoglobulin enhancer contains positive regulatory elements that activate transcription in B lymphocytes and negative regulatory elements that inhibit transcription in other cell types. The overall activity reflects the combined action of the proteins associated with each of the sequence elements.

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

68 Figure 7.21 Insulators

69 Regulation of Transcription in Eukaryotes Transcription factor binding sites have been identified by several types of experiments: Electrophoretic-mobility shift assay: Radiolabeled DNA fragments are incubated with a protein and then subjected to electrophoresis in a non- denaturing gel. Migration of a DNA fragment is slowed by a bound protein.

70 Figure 7.22 Electrophoretic-mobility shift assay

71 Regulation of Transcription in Eukaryotes Binding sites are usually short DNA sequences (6–10 base pairs) 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.

72 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.

73 Figure 7.23 Representative transcription factor binding sites

74 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.

75 Figure 7.24 Chromatin immunoprecipitation (Part 1)

76 Figure 7.24 Chromatin immunoprecipitation (Part 2)

77 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.

78 Key Experiment, Ch. 7, p. 259 (1)

79 Key Experiment, Ch. 7, p. 259 (2)

80 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.

81 Figure 7.25 Purification of Sp1 by DNA-affinity chromatography

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

83 Figure 7.26 Structure of transcriptional activators

84 Regulation of Transcription in Eukaryotes Many different 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.

85 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.

86 Figure 7.27 Examples of DNA-binding domains (Part 1)

87 Figure 7.27 Examples of DNA-binding domains (Part 2)

88 Regulation of Transcription in Eukaryotes 2. Helix-turn-helix domain: one helix makes most of the contacts with DNA, the other helices lie across the complex to stabilize the interaction. They include homeodomain proteins, important in the regulation of gene expression during embryonic development.

89 Regulation of Transcription in Eukaryotes Homeodomain proteins were first discovered as developmental mutants in Drosophila. They result in development of flies in which one body part is transformed into another. In Antennapedia, legs rather than antennae grow from the head.

90 Figure 7.28 The Antennapedia mutation (Part 1)

91 Figure 7.28 The Antennapedia mutation (Part 2)

92 Regulation of Transcription in Eukaryotes 3. Leucine zipper and helix-loop-helix proteins contain DNA-binding domains 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.

93 Figure 7.27 Examples of DNA-binding domains (Part 3)

94 Figure 7.27 Examples of DNA-binding domains (Part 4)

95 Regulation of Transcription in Eukaryotes The activation domains of transcription factors are not as well characterized as their DNA-binding domains. Activation domains stimulate transcription by two mechanisms: Interact with Mediator proteins and general transcription factors Interact with coactivators to modify chromatin structure.

96 Figure 7.30 Action of eukaryotic repressors

97 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.

98 Figure 7.30 Action of eukaryotic repressors (Part 1)

99 Regulation of Transcription in Eukaryotes Active repressors have specific 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.

100 Figure 7.30 Action of eukaryotic repressors (Part 2)

101 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.

102 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).

103 Figure 7.31 Regulation of transcriptional elongation (Part 1)

104 Figure 7.31 Regulation of transcriptional elongation (Part 2)

105 Regulation of Transcription in Eukaryotes The packaging of eukaryotic DNA in chromatin has important consequences for transcription, so chromatin structure is a critical aspect of gene expression. Actively transcribed genes are in relatively decondensed chromatin, which can be seen in polytene chromosomes of Drosophila.

106 Figure 7.32 Decondensed chromosome regions in Drosophila

107 Regulation of Transcription in Eukaryotes But actively transcribed genes remain bound to histones and packaged in nucleosomes. The tight winding of DNA around nucleosomes is a major obstacle to transcription. Chromatin can be altered by histone modifications and nucleosome rearrangements.

108 Regulation of Transcription in Eukaryotes Histone acetylation: The amino-terminal tail domains of core histones are rich in lysine and can be modified by acetylation. Transcriptional activators and repressors are associated with histone acetyltransferases (HAT) and deacetylases (HDAC), respectively.

109 Figure 7.33 Histone acetylation (Part 1)

110 Figure 7.33 Histone acetylation (Part 2)

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

112 Figure 7.34 Patterns of histone modification (Part 1)

113 Regulation of Transcription in Eukaryotes Different patterns of histone modification are found at promoters compared with enhancers. Example: distinct patterns of lysine-4 methylation are characteristic of enhancers and promoters.

114 Figure 7.34 Patterns of histone modification (Part 2)

115 Regulation of Transcription in Eukaryotes Histone modification 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 histone modification.

116 Figure 7.35 Epigenetic inheritance of histone modifications

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

118 Figure 7.36 Chromatin remodeling factors

119 Regulation of Transcription in Eukaryotes To facilitate elongation, elongation factors become associated with the phosphorylated C-terminal domain of RNA polymerase II. They include histone modifying enzymes and chromatin remodeling factors that transiently displace nucleosomes during transcription.

120 Regulation of Transcription in Eukaryotes Transcription can also be regulated by noncoding RNA molecules:  Small-interfering RNAs (siRNAs)  MicroRNAs (miRNAs).

121 Regulation of Transcription in Eukaryotes siRNAs repress transcription of target genes by inducing histone modifications that lead to chromatin condensation and formation of heterochromatin. In the yeast S. pombe, siRNAs direct formation of heterochromatin at centromeres.

122 Regulation of Transcription in Eukaryotes The siRNAs associate with RNA- induced transcriptional silencing (RITS) complex. RITS includes proteins that induce chromatin condensation and methylation of histone H3 lysine-9.

123 Figure 7.37 Regulation of transcription by siRNAs

124 Regulation of Transcription in Eukaryotes Long noncoding RNAs also regulate gene expression: X chromosome inactivation occurs during development when most genes on one X chromosome in female cells are inactivated. This compensates for the fact that females have twice as many copies of most X chromosome genes as males.

125 Regulation of Transcription in Eukaryotes Noncoding RNA transcribed from a regulatory gene, Xist, on the inactive X chromosome, binds to and coats this chromosome. This leads to chromatin condensation and conversion to heterochromatin.

126 Figure 7.38 X chromosome inactivation

127 Regulation of Transcription in Eukaryotes Recent sequencing research suggests there are many long noncoding RNAs (lncRNAs) transcribed from the human genome that are functional regulators of gene expression. lncRNAs associate with chromatin regulatory proteins, and may recruit chromatin modifying proteins to target genes.

128 Regulation of Transcription in Eukaryotes DNA methylation also controls transcription in eukaryotes: Methyl groups are added at the 5-carbon position of cytosines (C) that precede guanines (G) (CpG dinucleotides). This methylation is correlated with transcriptional repression.

129 Figure 7.39 DNA methylation

130 Regulation of Transcription in Eukaryotes Methylation is common in transposable elements; it plays a key role in suppressing their movement. DNA methylation also plays a role in X chromosome inactivation.

131 Regulation of Transcription in Eukaryotes DNA methylation is a 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.

132 Figure 7.40 Maintenance of methylation patterns

133 Regulation of Transcription in Eukaryotes DNA methylation plays a role in genomic imprinting: the 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 specifically methylated during the development of male, but not female, germ cells.

134 Figure 7.41 Genomic imprinting

135 RNA Processing and Turnover Bacterial mRNAs are used immediately for protein synthesis while still being transcribed. Other RNAs must be processed in various ways in both prokaryotic and eukaryotic cells. Regulation of processing provides another level of control of gene expression.

136 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.

137 Figure 7.42 Processing of ribosomal RNAs

138 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 is responsible for catalytic activity.

139 Figure 7.43 Processing of transfer RNAs (Part 1)

140 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.

141 Figure 7.43 Processing of transfer RNAs (Part 2)

142 RNA Processing and Turnover In eukaryotes, pre-mRNAs are extensively modified before export from the nucleus. Transcription and processing are coupled. The C-terminal domain (CTD) of RNA polymerase II plays a key role in coordinating these processes.

143 RNA Processing and Turnover The 5′ end of the transcript is modified by addition of a 7-methylguanosine cap. Enzymes responsible for capping are recruited to the phosphorylated CTD following initiation, and the cap is added after transcription of the first 20 to 30 nucleotides.

144 Figure 7.44 Processing of eukaryotic messenger RNAs

145 RNA Processing and Turnover At the 3′ end, a poly-A tail is added by polyadenylation. Signals for polyadenylation include a highly conserved hexanucleotide (AAUAAA in mammalian cells), and a G-U rich downstream sequence element.

146 Figure 7.45 Formation of the 3' ends of eukaryotic mRNAs

147 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.

148 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.

149 RNA Processing and Turnover In vitro systems were used to study splicing: A gene containing an intron 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.

150 Figure 7.46 In vitro splicing

151 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 A 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.

152 Figure 7.47 Splicing of pre-mRNA

153 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.

154 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).

155 Key Experiment, Ch. 7, p. 284 (1)

156 Key Experiment, Ch. 7, p. 284 (2)

157 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.

158 Figure 7.48 Assembly of the spliceosome (Part 1)

159 Figure 7.49 Binding of U1 snRNA to the 5' splice site

160 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 followed by excision of the intron.

161 Figure 7.48 Assembly of the spliceosome (Part 2)

162 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 can catalyze removal of their own introns in the absence of other protein or RNA factors.

163 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.

164 Figure 7.50 Self-splicing introns

165 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.

166 Figure 7.51 Role of splicing factors in spliceosome assembly

167 RNA Processing and Turnover Alternative splicing occurs frequently in genes of complex eukaryotes. Most pre-mRNAs have multiple introns, thus different mRNAs can be produced from the same gene. This provides a means of controlling gene expression, and increases the diversity of proteins that can be encoded.

168 RNA Processing and Turnover Sex determination in Drosophila is an example of tissue-specific alternative splicing. Alternative 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.

169 Figure 7.52 Alternative splicing in Drosophila sex determination

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

171 Figure 7.53 Alternative splicing of Dscam

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

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

174 Figure 7.54 Editing of apolipoprotein B mRNA

175 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.

176 RNA Processing and Turnover Aberrant 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.

177 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.

178 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.

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

180 RNA Processing and Turnover In eukaryotic cells, mRNA half-lives vary; less than 30 minutes to 20 hours in mammalian cells. 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.

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

182 Figure 7.55 mRNA degradation

183 RNA Processing and Turnover These RNA-binding proteins are regulated by extracellular signals, such as growth factors and hormones. Degradation of some mRNAs is regulated by both siRNAs and miRNA.


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