Regulation of Transcription in Eukaryotes

Transcription

1 Regulation of Transcription in Eukaryotes 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.

2 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.

3 Regulation of Transcription in Eukaryotes Gene expression in eukaryotic cells is also regulated by repressors which bind to specific DNA sequences and 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.

4 Regulation of Transcription in Eukaryotes Active repressors contain specific functional 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.

5 Regulation of Transcription in Eukaryotes Transcription can also be regulated at the level of elongation. Recent studies show that many genes in both Drosophila and humans are characterized by molecules of RNA polymerase II that have initiated transcription but are stalled immediately downstream of promoters. 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 transcriptionelongation factor-b).

6 Figure 7.32 Regulation of transcriptional elongation

7 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.

8 Regulation of Transcription in Eukaryotes But, actively transcribed genes remain bound to histones and packaged in nucleosomes. The tight winding of DNA around the nucleosome core particle is a major obstacle to transcription.

9 Regulation of Transcription in Eukaryotes Chromatin can be modified by: Interactions with HMG (high mobility group) proteins Modifications of histones Rearrangements of nucleosomes HMG proteins include: HMGA and HMGB, which can bend DNA and facilitate binding of regulatory factors to chromatin. HMGN binds to nucleosomes at sites that overlap the binding site of histone H1; they induce chromatin unfolding and maintain decondensed chromatin structure.

10 Regulation of Transcription in Eukaryotes Histone Modification 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.

11 Figure 7.34 Histone acetylation (Part 2)

12 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.

13 Regulation of Transcription in Eukaryotes Acetylated lysines serve as binding sites for many proteins that activate transcription. Other proteins associated with transcriptional activation bind to methylated lysine-4 residues. In contrast, methylation of lysines 9 and 27 is associated with repression and chromatin condensation. 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.

14 Figure 7.36 Epigenetic inheritance of histone modifications (Part 1)

15 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.

16 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. Transcription can also be regulated by noncoding RNA molecules, including small-interfering RNAs (sirnas) and micrornas (mirnas). They can induce histone modifications that lead to chromatin condensation and formation of heterochromatin.

17 Regulation of Transcription in Eukaryotes In the yeast S. pombe, sirnas direct formation of heterochromatin at centromeres, by associating with the RNA-induced transcriptional silencing (RITS) complex. RITS includes proteins that induce chromatin condensation and methylation of histone H3 lysine-9.

18 Regulation of Transcription in Eukaryotes X chromosome inactivation occurs during development, when most of the genes on one of the two X chromosomes in female cells are inactivated. This compensates for the fact that females have twice as many copies of most X chromosome genes as males. 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.

19 Regulation of Transcription in Eukaryotes DNA methylation is another general mechanism that controls transcription in eukaryotes. Methyl groups are added at the 5-carbon position of cytosines (C) that precede guanines (G) (CpG dinucleotides).

20 Regulation of Transcription in Eukaryotes Methylation is common in transposable elements, it plays a key role in suppressing the movement of transposons. DNA methylation is associated with transcriptional repression of some genes, and also has a role in X chromosome inactivation. DNA methylation is a mechanism of epigenetic inheritance. Following DNA replication, an enzyme methylates CpG sequences of a daughter strand that is hydrogenbonded to a methylated parental strand.

21 Figure 7.41 Maintenance of methylation patterns

22 Regulation of Transcription in Eukaryotes DNA methylation also plays a role in genomic imprinting: expression depends on whether they are inherited from the mother or from 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.

23 RNA Processing and Turnover Most newly-synthesized RNAs must be modified, except bacterial RNAs which are used immediately for protein synthesis while still being transcribed. rrnas and trnas must be processed in both prokaryotic and eukaryotic cells. Regulation of processing steps provides another level of control of gene expression.

24 RNA Processing and Turnover The ribosomal RNAs of both prokaryotes and eukaryotes are derived from a single long pre-rrna molecule. 5S rrna in eukaryotes is transcribed from a separate gene.

25 RNA Processing and Turnover trnas also start as long precursor molecules (pre-trnas), in both 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.

26 Figure 7.44 Processing of transfer RNAs (Part 2)

27 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. 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.

28 Figure 7.45 Processing of eukaryotic messenger RNAs (Part 1)

29 RNA Processing and Turnover At the 3 end, a poly-a tail is added by a process called polyadenylation. The signals for polyadenylation include a highly conserved hexanucleotide (AAUAAA in mammalian cells), and a G-U rich downstream sequence element.

30 RNA Processing and Turnover Cleavage and polyadenylation are followed by degradation of the RNA that has been synthesized downstream, resulting in termination of transcription. Introns (noncoding sequences) are also removed from pre-mrna by splicing. In vitro systems were necessary to understand splicing: a gene containing an intron is cloned downstream of a promoter recognized by a bacteriophage RNA polymerase.

31 RNA Processing and Turnover In vitro transcription of the plasmid yields pre-mrnas. When these are added to nuclear extracts of mammalian cells, splicing occurs. The system allows splicing to be analyzed in greater detail than in intact cells.

32 Figure 7.47 In vitro splicing (Part 1)

33 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 (the branch point). The intron forms a loop. 2. Cleavage at the 3 splice site and simultaneous ligation of the exons, resulting in excision of the intron loop.

34 RNA Processing and Turnover Three sequence elements of premrnas are important: Sequences 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.

35 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. They are complexed with six to ten protein molecules to form small nuclear ribonucleoprotein particles (snrnps).

36 RNA Processing and Turnover The first step in spliceosome assembly is the binding of U1 snrnp to the 5 SS. The recognition of 5 SS involves base pairing between the 5 SS consensus sequence and a complementary sequence at the 5 end of U1 snrna.

37 Figure 7.50 Binding of U1 snrna to the 5 splice site

38 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.

39 RNA Processing and Turnover snrnas recognize consensus sequences 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. Two groups of self-splicing introns: Group I cleavage at 5 SS mediated by a guanosine cofactor. The 3 end of the free exon then reacts with the 3 SS to excise the intron. Group II cleavage of 5 SS results from attack by an adenosine nucleotide in the intron resulting in a lariat-like intermediate, which is excised.

40 Figure 7.51 Self-splicing introns

41 RNA Processing and Turnover Other splicing factors bind to specific RNA sequences and recruit U1 and U2 snrnps to the appropriate sites on pre-mrna. SR 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.

42 RNA Processing and Turnover Alternative splicing occurs frequently in genes of complex eukaryotes. Because most pre-mrnas contain multiple introns, different mrnas can be produced from the same gene. This provides a novel means of controlling gene expression, and increasing the diversity of proteins that can be encoded. Sex determination in Drosophila is an example of tissuespecific alternative splicing. Alternative splicing of transformer mrna is regulated by the SXL protein, which is only expressed in female flies. SXL acts as a repressor that blocks splicing factor U2AF.

43 Figure 7.53 Alternative splicing in Drosophila sex determination

44 RNA Processing and Turnover The Dscam gene of Drosophila contains four sets of alternative exons, one from each set goes into the spliced mrna. The exons can be joined in any combination, so alternative splicing can potentially yield 38,016 different mrnas.

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

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

47 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. 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.

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

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

50 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. 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. 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.

51 Figure 7.56 mrna degradation