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Bio 108 Cell Biology lec 6 Regulation of Transcription Initiation

Gene expression
Transcription in Prokaryotes

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Bio 108 Cell Biology lec 6 Regulation of Transcription Initiation

  1. 1. Regulation of Transcription Initiation Gene expression -overall process by which the information encoded in a gene is converted into an observable phenotype (most commonly production of a protein). Why- regulate gene expression? • To adjust to sudden changes • To conserve energy • To save resources Gene control- All of the mechanisms involved in regulating gene expression. Most common is regulation of transcription, although mechanisms influencing the processing, stabilization, and translation of mRNAs help control expression of some genes.
  2. 2. TYPE OF RNA FUNCTION mRNAs messenger RNAs, code for proteins rRNAs ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis tRNAs transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids snRNAs small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA snoRNAs small nucleolar RNAs, used to process and chemically modify rRNAs Other noncoding RNAs function in diverse cellular processes, including telomere synthesis, X-chromosome inactivation, and the transport of proteins into the ER Table 6-1. Principal Types of RNAs Produced in Cells
  3. 3. Regulation of gene expression allows cells to adapt to changes in their environments and is responsible for the distinct activities of the multiple differentiated cell types that make up complex plants and animals. Control of transcription initiation—the first step—is the most important mechanism for determining whether or not most genes are expressed and how much of the encoded mRNAs, and consequently proteins, are produced. Transcription - a process in which a DNA strand provides the information for the synthesis of an RNA strand. -enzymes responsible for transcription in both prokaryotic and eukaryotic cells are called DNA-dependent RNA polymerases or simply RNA polymerases.
  4. 4. Transcription in Prokaryotes RNA polymerase- An enzyme that catalyzes the synthesis of RNA. -the complete enzyme consists of five subunits: two α, one β, one β′, and one σ. -catalyzes the growth of RNA chains always in the 5′ to 3′ direction (similar to that of DNA pol) - however, RNA polymerase does not require a preformed primer to initiate the synthesis of RNA. -σ subunit is required to identify the correct sites for transcription initiation
  5. 5. Promoter -a DNA sequence to which RNA polymerase binds to initiate transcription. Figure 6.2. Sequences of E. coli promoters E. coli promoters are characterized by two sets of sequences located 10 and 35 base pairs upstream of the transcription start site (+1). The consensus sequences shown correspond to the bases most frequently found in different promoters. *role of σ is to direct the polymerase to promoters by binding specifically to both the -35 and -10 sequences, leading to the initiation of transcription at the beginning of a gene Pribnow box
  6. 6. Figure 6.4. Transcription by E. coli RNA polymerase a. the polymerase initially binds nonspecifically to DNA and migrates along the molecule until the σ subunit binds to the -35 and -10 promoter elements, forming a closed-promoter complex. b. the polymerase then unwinds (15 bases) DNA around the initiation site c. transcription is initiated by the polymerization of 2 free NTPs. d. σ subunit then dissociates from the core polymerase, which migrates along the DNA and elongates the growing RNA chain.
  7. 7. RNA Chain Termination - Termination of RNA chain synthesis appears to be brought about by two types of mechanisms. In the first type the termination signal appears to be recognized by DNA itself. RNA polymerase reads an extended poly (A) sequence on DNA. This results in an RNA transcript with a terminal poly(U) sequence The second type of termination signal involves an additional protein called the rho (P) factor. Rho factor is an essential transcription protein in prokaryotes. In Escherichia coli, it is a ~275 kD hexamer of identical subunits. Each subunit has an RNA-binding domain and an ATP-hydrolysis domain. Rho is a member of the family of ATP-dependent hexameric helicases that function by passing nucleic acids through the hole in the middle of the hexamer. Rho functions as an ancillary factor for RNA polymerase. Rho- dependent terminators account for about half of E. coli terminators. The (rho) factor probably binds to RNA polymerase. It is however, not certain whether it also, or exclusively, binds to DNA.
  8. 8. 1. Intrinsic termination (also called Rho-independent termination) is a mechanism in both eukaryotes and prokaryotes that causes mRNA transcription to be stopped. In this mechanism, the mRNA contains a sequence that can base pair with itself to form a stem-loop structure 7-20 base pairs in length that is also rich in Cytosine-Guanine base pairs. Figure 6.5. Transcription termination The termination of transcription is signaled by a GC-rich inverted repeat followed by four A residues. The inverted repeat forms a stable stem-loop structure in the RNA, causing the RNA to dissociate from the DNA template.
  9. 9. 2. Rho Dependent Polarity In Prokaryotes transcription and translation are coupled -- that is, ribosomes begin translation while the mRNA is being produced. Coupled transcription and translation. During transcription RNA polymerase (RNAP) separates the two DNA strands and forms a short region of base pairing between the coding strand of DNA and the newly synthesized RNA. As the RNA elongates it is displaced as the single-stranded DNA rehybridizes to form double-stranded DNA. Ribosomes rapidly bind to the resulting single-stranded RNA and begin protein synthesis. Rho dependent polarity. The process of Rho dependent polarity is shown stepwise .Rho dependent polarity is usually initiated by premature transcription termination. When ribosomes encounter a stop (aka "nonsense") codon, they fall off of the RNA.
  10. 10. Rho factor then interacts with RNAP, causing it to fall off of the DNA which terminates transcription. Thus, when translation termination occurs within a gene it can cause transcriptional termination, preventing expression of downstream genes. This process is called Rho dependent polarity is clearly more complex than the model shown above. There is biochemical and genetic evidence that Rho factor interacts with other transcription termination factors (for example, Nus) and the alpha subunit of RNAP. Rho factor cannot bind to RNA coated with ribosomes, but Rho factor binds to specific regions in naked RNA.
  11. 11. Concept 1: Gene Regulation in Bacteria Bacteria adapt to changes in their surroundings by using regulatory proteins to turn groups of genes on and off in response to various environmental signals. The DNA of Escherichia coli is sufficient to encode about 4000 proteins, but only a fraction of these are made at any one time. E. coli regulates the expression of many of its genes according to the food sources that are available to it.
  12. 12. Concept 2: The Lactose Operon (Jacob-Monod model) -An operon is a cluster of bacterial genes along with an adjacent promoter that controls the transcription of those genes. When the genes in an operon are transcribed, a single mRNA is produced for all the genes in that operon. This mRNA is said to be polycistronic because it carries the information for more than one type of protein.
  13. 13. An operon • Promoter (DNA) – where RNA polymerase binds • Operator (DNA) – lies between promoter and structural genes (regulatory sequence of DNA that controls transcription of an operon) •Repressor (protein) – binds to operator to block transcription
  14. 14. Concept 3: The lac Operator The operator is a short region of DNA that lies partially within the promoter and that interacts with a regulatory protein that controls the transcription of the operon. *Here's an analogy. A promoter is like a doorknob, in that the promoters of many operons are similar. An operator is like the keyhole in a doorknob,in that each door is locked by only a specific key, which in this analogy is a specific regulatory protein.
  15. 15. The lac operon of E.coli – an inducible system includes 3 genes: 1. lacZ –encodes β-galactosidase (catalyzes the hydrolysis of lactose to glucose and galactose) 2. lacY – encodes lactose permease (carrier protein in the bacterial plasma membrane that moves the sugar into the cells) 3. lacA- encodes thiogalactoside transacetylase (transfers acetyl group from acetyl-CoA to β-galactosides)
  16. 16. Figure 10-2. Jacob and Monod model of transcriptional regulation of the lac operon by lac repressor. 1. When lac repressor binds to a DNA sequence called the operator (O), which lies just upstream of the lacZ gene, transcription of the operon by RNA polymerase is blocked. 2. 2. Binding of lactose to the repressor causes a conformational change in the repressor, so that it no longer binds to the operator. RNA polymerase then is free to bind to the promoter (P) and initiate transcription of the lac genes; 3. the resulting polycistronic mRNA is translated into the encoded proteins.
  17. 17. Concept 4: The lac Regulatory Gene The regulatory gene lacI produces an mRNA that produces a Lac repressor protein, which can bind to the operator of the lac operon. In some texts, the lacI regulatory gene is called the lacI regulator gene. Regulatory genes are not necessarily close to the operons they affect. The general term for the product of a regulatory gene is a regulatory protein. The Lac regulatory protein is called a repressor because it keeps RNA polymerase from transcribing the structural genes. Thus the Lac repressor inhibits transcription of the lac operon.
  18. 18. Concept 5: The Lac Repressor Protein In the absence of lactose, the Lac repressor binds to the operator and keeps RNA polymerase from transcribing the lac genes. It would be energetically wasteful for E. coli if the lac genes were expressed when lactose was not present.
  19. 19. Concept 6: The Effect of Lactose on the lac Operon When lactose is present, the lac genes are expressed because allolactose binds to the Lac repressor protein and keeps it from binding to the lac operator. Allolactose is an isomer of lactose. Small amounts of allolactose are formed when lactose enters E. coli. Allolactose binds to an allosteric site on the repressor protein causing a conformational change. As a result of this change, the repressor can no longer bind to the operator region and falls off. RNA polymerase can then bind to the promoter and transcribe the lac genes.
  20. 20. Concept 7: The lac Inducer: Allolactose Allolactose is called an inducer because it turns on, or induces the expression of, the lac genes. The presence of lactose (and thus allolactose) determines whether or not the Lac repressor is bound to the operator.
  21. 21. Allolactose binds to an allosteric site on the repressor protein causing a conformational change. As a result of this change, the repressor can no longer bind to the operator region and falls off. RNA polymerase can then bind to the promoter and transcribe the lac genes.
  22. 22. Concept 8: Feedback Control of the lac Operon When the enzymes encoded by the lac operon are produced, they break down lactose and allolactose, eventually releasing the repressor to stop additional synthesis of lac mRNA. Messenger RNA breaks down after a relatively short amount of time.
  23. 23. Concept 9: Energy Source Preferences of E. coli Whenever glucose is present, E. coli metabolizes it before using alternative energy sources such as lactose, arabinose, galactose, and maltose. Glucose is the preferred and most frequently available energy source for E. coli. The enzymes to metabolize glucose are made constantly by E. coli. When both glucose and lactose are available, the genes for lactose metabolism are transcribed at low levels. Only when the supply of glucose has been exhausted does does RNA polymerase start to transcribe the lac genes efficiently, which allows E. coli to metabolize lactose.
  24. 24. Concept 10: The Effect of Glucose and Lactose on the lac Operon When both glucose and lactose are present, the genes for lactose metabolism are transcribed to a small extent.
  25. 25. Maximal transcription of the lac operon occurs only when glucose is absent and lactose is present. The action of cyclic AMP and a catabolite activator protein produce this effect. Positive Control and Catabolite Repression
  26. 26. Concept 11: The Effect of Glucose and Cyclic AMP on the lac Operon The presence or absence of glucose affects the lac operon by affecting the concentration of cyclic AMP. *The concentration of cyclic AMP in E. coli is inversely proportional to the concentration of glucose: as the concentration of glucose decreases, the concentration of cyclic AMP increases. Cyclic AMP is derived from ATP.
  27. 27. Concept 12: The Effect of Lactose in the Absence of Glucose on the lac Operon In the presence of lactose and absence of glucose, cyclic AMP (cAMP) joins with a catabolite activator protein that binds to the lac promoter and facilitates the transcription of the lac operon. In some texts, the catabolite activator protein (CAP) is called the cAMP-receptor protein. When the concentration of glucose is low, cAMP accumulates in the cell. The binding of cAMP and the catabolite activator protein to the lac promoter increases transcription by enhancing the binding of RNA polymerase to the lac promoter.
  28. 28. The lac operon – summary • In the absence of inducer, the operon is turned off • Control is exerted by a regulatory protein- the repressor- that turns the operon off. • Regulatory genes produce proteins whose sole function is to regulate the expression of other genes. • Certain other DNA sequences (operators and promoters) do not code for proteins, but are binding sites for regulatory or other proteins. • Adding inducers turns the operon on. Operator-repressor control that induces transcription.
  29. 29. The trp operon of E. coli – a repressible system • In repressible systems, the repressor protein cannot shut off its operon unless it first binds to a corepressor, which may be either the metabolic end product itself (tryptophan in this case) or an analog of it. Operator-repressor control that represses transcription
  30. 30. 1. Regulatory gene r produces an inactive repressor, which cannot bind to the operator mRNA Inactive repressor PTrp o e d c b a RNA polymerase Transcription proceeds DNA mRNA transcript Translation e d c b a Tryptophan absent DNA r Enzymes of the tryptophan Synthesis pathway 2. RNA polymerase transcribes the structural genes. Translation makes the enzymes of the tryptophan synthesis pathway.
  31. 31. mRNA Inactive repressor PTrp o e d c b a DNA Tryptophan present DNA r Corepressor (tryptophan) Active repressor 1. Tryptophan binds the repressor… 2. … which then binds to the operator. 3. Trp blocks RNA pol from binding and transcribing the structural genes, preventing synthesis of trp pathway enzymes
  32. 32. Inducible vs. Repressible systems • in inducible systems, the substrate of a metabolic pathway (the inducer) interacts with a regulatory protein (the repressor) to render it incapable of binding to the operator, thus allowing transcription • in repressible systems, the product of a metabolic pathway (the corepressor) interacts with the regulatory protein to make it capable of binding to the operator, thus blocking the transcription • In both kinds of systems, the regulatory molecule functions by binding to the operator.
  33. 33. Examples of Negative control of transcription: a. inducible lac system b. repressible trp system •The two operator-repressor systems because the regulatory molecule (the repressor) in each case prevents transcription. Example of Positive control of transcription: a. promoter-catabolite repression system • because the regulatory molecule (the CRP-cAMP complex) enhances transcription.
  34. 34. Transcription in Eukaryotes Transcription Initiation in Eucaryotes Requires Many Proteins - eucaryotic nuclei have three RNA pol (bacteria only one): a. RNA polymerase I b. RNA polymerase II c. and RNA polymerase III * structurally similar to one another but they transcribe different types of genes TYPE OF POLYMERASE GENES TRANSCRIBED RNA polymerase I 5.8S, 18S, and 28S rRNA genes RNA polymerase II all protein-coding genes, plus snoRNAgenes and some snRNA genes RNA polymerase III tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs Table 6-2. The Three RNA Polymerases in Eucaryotic Cells
  35. 35. Transcription factor (TF) - General term for any protein, other than RNA polymerase, required to initiate or regulate transcription in eukaryotic cells. General factors, required for transcription of all genes, participate in formation of the transcription-initiation complex near the start site. Specific factors stimulate (or repress) transcription of particular genes by binding to their regulatory sequences.
  36. 36. Several important differences in the way in which the bacterial and eucaryotic enzymes function: 1. While bacterial RNA polymerase (with σ factor as one of its subunits) is able to initiate transcription on a DNA template in vitro without the help of additional proteins, eucaryotic RNA polymerases cannot. They require the help of a large set of proteins called general transcription factors, which must assemble at the promoter with the polymerase before the polymerase can begin transcription. 2. Eucaryotic transcription initiation must deal with the packing of DNA into nucleosomes and higher order forms of chromatin structure, features absent from bacterial chromosomes. RNA Polymerase II Requires General Transcription Factors General transcription factor -Any of the proteins whose assembly around the TATA box is required for the initiation of transcription of most eucaryotic genes. TATA box -Consensus sequence in the promoter region of many eucaryotic genes that binds a general transcription factor and hence specifies the position at which transcription is initiated.
  37. 37. TATA-binding protein (TBP) -A basal transcription factor that binds directly to the TATA box. TFIID (Transcription factorIID) is itself composed of multiple subunits, including the TATA-binding protein (TBP), which binds specifically to the TATAA consensus sequence, and 10-12 other polypeptides, called TBP-associated factors (TAFs) Figure 6.14. RNA polymerase II holoenzyme The holoenzyme consists of a preformed complex of RNA polymerase II, the general transcription factors TFIIB, TFIIE, TFIIF, and TFIIH, and several other proteins that activate transcription. This complex can be recruited directly to a promoter via interaction with TFIID (TBP + TAFs).
  38. 38. Figure 6-16. Initiation of transcription of a eucaryotic gene by RNA polymerase II. To begin transcription, RNA polymerase requires a number of general transcription factors (called TFIIA, TFIIB, and so on). (A) The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site at which transcription is initiated. (B) The TATA box is recognized and bound by transcription factor TFIID, which then enables the adjacent binding of TFIIB (C). For simplicity the DNA distortion produced by the binding of TFIID (see Figure 6-18) is not shown. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then uses ATP to pry apart the DNA double helix at the transcription start point, allowing transcription to begin. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription.
  39. 39. TFIIH is a multisubunit factor that appears to play at least two important roles: a. First, two subunits of TFIIH are helicases, which may unwind DNA around the initiation site. (These subunits of TFIIH are also required for nucleotide excision repair). b. Another subunit of TFIIH is a protein kinase that phosphorylates repeated sequences present in the C-terminal domain of the largest subunit of RNA polymerase II. Phosphorylation of these sequences is thought to release the polymerase from its association with the initiation complex, allowing it to proceed along the template as it elongates the growing RNA chain
  40. 40. Figure 6.12. Formation of a polymerase II transcription complex Many polymerase II promoters have a TATA box (consensus sequence TATAA) 25 to 30 nucleotides upstream of the transcription start site. This sequence is recognized by transcription factor TFIID, which consists of the TATA-binding protein (TBP) and TBP- associated factors (TAFs). TFIIB(B) then binds to TBP, followed by binding of the polymerase in association with TFIIF(F). Finally, TFIIE(E) and TFIIH(H) associate with the complex.
  41. 41. Figure 10-6. DNase I footprinting, a common technique for identifying protein-binding sites in DNA. a. A DNA fragment is labeled at one end with 32P (red dot) as in the Maxam-Gilbert sequencing method. b. Portions of the sample then are digested with DNase I in the presence and absence of a protein that binds to a specific sequence in the fragment. (Bottom) Diagram of hypothetical autoradiogram of the gel for the minus protein sample above reveals bands corresponding to all possible fragments produced by DNase I cleavage (−lane). In the sample digested in the presence of a DNA- binding protein, two bands are missing (+lane); these correspond to the DNA region protected from digestion by bound protein and are referred to as the footprint of that protein.
  42. 42. Polymerase II Also Requires Activator, Mediator, and Chromatin- modifying Proteins DNA in eucaryotic cells is packaged into nucleosomes, which are further arranged in higher-order chromatin structures. As a result, transcription initiation in a eucaryotic cell is more complex and requires more proteins 1. gene regulatory proteins known as transcriptional activators bind to specific sequences in DNA and help to attract RNA polymerase II to the start point of transcription (This attraction is needed to help the RNA polymerase and the general transcription factors in overcoming the difficulty of binding to DNA that is packaged in chromatin). 2. eucaryotic transcription initiation in vivo requires the presence of a protein complex known as the mediator, which allows the activator proteins to communicate properly with the polymerase II and with the general transcription factors. 3. transcription initiation in the cell often requires the local recruitment of chromatin-modifying enzymes, including chromatin remodeling complexes and histone acetylases (both types of enzymes can allow greater accessibility to the DNA present in chromatin, and so, they facilitate the assembly of the transcription initiation machinery onto DNA).
  43. 43. A typical eucaryotic gene has many activator proteins, which together determine its rate and pattern of transcription. Sometimes acting from a distance of several thousand nucleotide pairs (indicated by the dashed DNA molecule), these gene regulatory proteins help RNA polymerase, the general factors, and the mediator all to assemble at the promoter. *activators attract ATP- dependent chromatin- remodeling complexes and histone acetylases.
  44. 44. Protein-coding genes have •exons whose sequence encodes the polypeptide; •introns that will be removed from the mRNA before it is translated •a transcription start site •a promoter the basal or core promoter located within about 40 bp of the start site an "upstream" promoter, which may extend over as many as 200 bp farther upstream •enhancers •silencers Adjacent genes (RNA-coding as well as protein-coding) are often separated by an insulator which helps them avoid cross- talk between each other's promoters and enhancers (and/or silencers).
  45. 45. Enhancers Some transcription factors ("Enhancer-binding protein") bind to regions of DNA that are thousands of base pairs away from the gene they control. Binding increases the rate of transcription of the gene. Enhancers can be located upstream, downstream, or even within the gene they control. How does the binding of a protein to an enhancer regulate the transcription of a gene thousands of base pairs away? One possibility is that enhancer-binding proteins — in addition to their DNA- binding site, have sites that bind to transcription factors ("TF") assembled at the promoter of the gene. This would draw the DNA into a loop (as shown in the figure).
  46. 46. Visual evidence Michael R. Botchan (who kindly supplied these electron micrographs) and his colleagues have produced visual evidence of this model of enhancer action. They created an artificial DNA molecule with •several (4) promoter sites for Sp1 about 300 bases from one end. Sp1 is a zinc-finger transcription factor that binds to the sequence 5' GGGCGG 3' found in the promoters of many genes, especially "housekeeping" genes. •several (5) enhancer sites about 800 bases from the other end. These are bound by an enhancer- binding protein designated E2. •1860 base pairs of DNA between the two. When these DNA molecules were added to a mixture of Sp1 and E2, the electron microscope showed that the DNA was drawn into loops with "tails" of approximately 300 and 800 base pairs. At the neck of each loop were two distinguishable globs of material, one representing Sp1 (red), the other E2 (blue) molecules. (The two micrographs are identical; the lower one has been labeled to show the interpretation.) Artificial DNA molecules lacking either the promoter sites or the enhancer sites, or with mutated versions of them, failed to form loops when mixed with the two proteins.
  47. 47. Silencers Silencers are control regions of DNA that, like enhancers, may be located thousands of base pairs away from the gene they control. However, when transcription factors bind to them, expression of the gene they control is repressed. Insulators A problem: As you can see above, enhancers can turn on promoters of genes located thousands of base pairs away. What is to prevent an enhancer from inappropriately binding to and activating the promoter of some other gene in the same region of the chromosome? One answer: an insulator. Insulators are •stretches of DNA (as few as 42 base pairs may do the trick) •located between the enhancer(s) and promoter or silencer(s) and promoter of adjacent genes or clusters of adjacent genes. Their function is to prevent a gene from being influenced by the activation (or repression) of its neighbors.
  48. 48. Example: The enhancer for the promoter of the gene for the delta chain of the gamma/delta T-cell receptor for antigen (TCR) is located close to the promoter for the alpha chain of the alpha/beta TCR (on chromosome 14 in humans). A T cell must choose between one or the other. There is an insulator between the alpha gene promoter and the delta gene promoter that ensures that activation of one does not spread over to the other.
  49. 49. All insulators discovered so far in vertebrates work only when bound by a protein designated CTCF ("CCCTC binding factor"; named for a nucleotide sequence found in all insulators). CTCF has 11 zinc fingers. Another example: In mammals (mice, humans, pigs), only the allele for insulin- like growth factor-2 (IGF2) inherited from one's father is active; that inherited from the mother is not — a phenomenon called imprinting. The mechanism: the mother's allele has an insulator between the IGF2 promoter and enhancer. So does the father's allele, but in his case, the insulator has been methylated. CTCF can no longer bind to the insulator, and so the enhancer is now free to turn on the father's IGF2 promoter. Many of the commercially-important varieties of pigs have been bred to contain a gene that increases the ratio of skeletal muscle to fat. This gene has been sequenced and turns out to be an allele of IGF2, which contains a single point mutation in one of its introns. Pigs with this mutation produce higher levels of IGF2 mRNA in their skeletal muscles (but not in their liver). This tells us that: •Mutations need not be in the protein-coding portion of a gene in order to affect the phenotype. •Mutations in non-coding portions of a gene can affect how that gene is regulated (here, a change in muscle but not in liver).
  50. 50. Transcription by RNA Polymerases I and III All three RNA polymerases, however, require additional transcription factors to associate with appropriate promoter sequences. Furthermore, although the three different polymerases in eukaryotic cells recognize distinct types of promoters, a common transcription factor—the TATA- binding protein (TBP)—appears to be required for initiation of transcription by all three enzymes. RNA polymerase I is devoted solely to the transcription of ribosomal RNA genes, which are present in tandem repeats. Figure 6.15. The ribosomal RNA gene The ribosomal DNA (rDNA) is transcribed to yield a large RNA molecule (45S pre- rRNA), which is then cleaved into 28S, 18S, and 5.8S rRNAs.
  51. 51. Figure 6.16. Initiation of rDNA transcription Two transcription factors, UBF and SL1, bind cooperatively to the rDNA promoter and recruit RNA polymerase I to form an initiation complex. One subunit of SL1 is the TATA-binding protein (TBP). UBF (upstream binding factor) SL1 (selectivity factor 1)
  52. 52. -promoter for ribosomal RNA genes does not contain a TATA box, TBP does not bind to specific promoter sequences. Instead, the association of TBP with ribosomal RNA genes is mediated by the binding of other proteins in the SL1 complex to the promoter, a situation similar to the association of TBP with the Inr sequences of polymerase II genes that lack TATA boxes. -genes for tRNAs, 5S rRNA, and some of the small RNAs involved in splicing and protein transport are transcribed by polymerase III. -these genes are characterized by promoters that lie within, rather than upstream of, the transcribed sequence
  53. 53. Figure 6.17. Transcription of polymerase III genes The promoters of 5S rRNA and tRNA genes are downstream of the transcrip-tion initiation site. Transcription of the 5S rRNA gene is initiated by the binding of TFIIIA, followed by the binding of TFIIIC, TFIIIB, and the polymerase. The tRNA promoters do not contain a binding site for TFIIIA, and TFIIIA is not required for their transcription. Instead, TFIIIC initiates the transcription of tRNA genes by binding to promoter sequences, followed by the association of TFIIIB and polymerase. The TATA-binding protein (TBP) is a subunit of TFIIIB.
  54. 54. RNA Processing and Turnover Most newly synthesized RNAs must be modified in various ways to be converted to their functional forms except bacterial mRNAs . -the primary transcripts of both rRNAs and tRNAs must undergo a series of processing steps in prokaryotic as well as eukaryotic cells. Processing of Ribosomal and Transfer RNAs basic processing of rRNA and tRNAs in prokaryotic and eukaryotic cells is similar, as might be expected given the fundamental roles of these RNAs in protein synthesis. *eukaryotes have four species of ribosomal RNAs, three of which (the 28S, 18S, and 5.8S rRNAs) are derived by cleavage of a single long precursor transcript, called a pre-rRNA *prokaryotes have three ribosomal RNAs (23S, 16S, and 5S), which are equivalent to the 28S, 18S, and 5S rRNAs of eukaryotic cells and are also formed by the processing of a single pre-rRNA transcript.
  55. 55. pre-rRNA -The primary transcript, which is cleaved to form individual ribosomal RNAs (the 28S, 18S, and 5.8S rRNAs of eukaryotic cells). Figure 6.37. Processing of ribosomal RNAs Prokaryotic cells contain three rRNAs (16S, 23S, and 5S), which are formed by cleavage of a pre-rRNA transcript. Eukaryotic cells (e.g., human cells) contain four rRNAs. One of these (5S rRNA) is transcribed from a separate gene; the other three (18S, 28S, and 5.8S) are derived from a common pre-rRNA. Following cleavage, the 5.8S rRNA (which is unique to eukaryotes) becomes hydrogen-bonded to 28S rRNA.
  56. 56. Figure 6.38. Processing of transfer RNAs (A) Transfer RNAs are derived from pre- tRNAs, some of which contain several individual tRNA molecules. Cleavage at the 5′ end of the tRNA is catalyzed by the RNase P ribozyme; cleavage at the 3′ end is catalyzed by a conventional protein RNase. A CCA terminus is then added to the 3′ end of many tRNAs in a posttranscriptional processing step. Finally, some bases are modified at characteristic positions in the tRNA molecule. In this example, these modified nucleosides include dihydrouridine (DHU), methylguanosine (mG), inosine (I), ribothymidine (T), and pseudouridine (y). (B) Structure of modified bases. Ribothymidine, dihydrouridine, and pseudouridine are formed by modification of uridines in tRNA. Inosine and methylguanosine are formed by the modification of guanosines. tRNAs in both bacteria and eukaryotes are synthesized as longer precursor molecules (pre-tRNAs), some of which contain several individual tRNA sequences pre-tRNA The primary transcript, which is cleaved to form transfer RNAs. In bacteria, some tRNAs are included in the pre-rRNA transcripts.
  57. 57. RNase P - A ribozyme that cleaves the 5´ end of pre-tRNAs. - consists of RNA and protein molecules, both of which are required for maximal activity. ribozyme An RNA enzyme. Note: an unusual aspect of tRNA processing is the extensive modification of bases in tRNA molecules. Approximately 10% of the bases in tRNAs are altered to yield a variety of modified nucleotides at specific positions in tRNA molecules (see Figure 6.38). The functions of most of these modified bases are unknown, but some play important roles in protein synthesis by altering the base-pairing properties of the tRNA molecule
  58. 58. Transcription Elongation in Eucaryotes Is Tightly Coupled To RNA Processing Figure 6-21. Summary of the steps leading from gene to protein in eucaryotes and bacteria. The final level of a protein in the cell depends on the efficiency of each step and on the rates of degradation of the RNA and protein molecules. (A) In eucaryotic cells the RNA molecule produced by transcription alone (sometimes referred to as the primary transcript) would contain both coding (exon) and noncoding (intron) sequences. Before it can be translated into protein, the two ends of the RNA are modified, the introns are removed by an enzymatically catalyzed RNA splicing reaction, and the resulting mRNA is transported from the nucleus to the cytoplasm.
  59. 59. Figure 6-23. The “RNA factory” concept for eucaryotic RNA polymerase II. Not only does the polymerase transcribe DNA into RNA, but it also carries pre-mRNA- processing proteins on its tail, which are then transferred to the nascent RNA at the appropriate time. There are many RNA-processing enzymes, and not all travel with the polymerase. For RNA splicing, for example, only a few critical components are carried on the tail; once transferred to an RNA molecule, they serve as a nucleation site for the remaining components. The RNA- processing proteins first bind to the RNA polymerase tail when it is phosphorylated late in the process of transcription initiation). Once RNA polymerase II finishes transcribing, it is released from DNA, the phosphates on its tail are removed by soluble phosphatases, and it can reinitiate transcription. Only this dephosphorylated form of RNA polymerase II is competent to start RNA synthesis at a promoter.