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Transcription and splicing.

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  • 1. Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 12 GENE TRANSCRIPTION AND RNA MODIFICATION (processing)
  • 2. The central dogma of genetics Figure 12.1 12-5
  • 3.
    • A key concept is that DNA base sequences define the beginning and end of a gene and regulate the level of RNA synthesis
    • Gene expression is the overall process by which the information within a gene is used to produce a functional product which can determine a trait in play with the environment
    12.1 OVERVIEW OF TRANSCRIPTION Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-6
  • 4. Figure 12.2 12-7 Signals the end of protein synthesis
  • 5. Gene Expression Requires Base Sequences Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The strand that is actually transcribed (used as the template) is termed the template strand
    • The opposite strand is called the coding strand or the sense strand
      • The base sequence is identical to the RNA transcript
        • Except for the substitution of uracil in RNA for thymine in DNA
    • Transcription factors recognize the promoter and regulatory sequences to control transcription
    • mRNA sequences such as the ribosomal-binding site and codons direct translation
    12-8
  • 6. The Stages of Transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Transcription occurs in three stages
      • Initiation
      • Elongation
      • Termination
    • These steps involve protein-DNA interactions
      • Proteins such as RNA polymerase interact with DNA sequences
      • Transcription factors that control transcription bind directly or indirectly to DNA
    12-9
  • 7. 12-10 Figure 12.3
  • 8. RNA Transcripts Have Different Functions Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Once they are made, RNA transcripts play different functional roles
    • Well over 90% of all genes are structural genes producing mRNA
    • The other RNA molecules are never translated: This collection appears much greater that initially believed; Some RNAs are 20-25 nts long that have important functions!
    12-11
  • 9. RNA Transcripts Have Different Functions Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The RNA transcripts from nonstructural genes are not translated
      • They do have various important cellular functions
      • They can still confer traits
      • In some cases, the RNA transcript becomes part of a complex that contains protein subunits
        • For example
          • Ribosomes
          • Spliceosomes
          • Signal recognition particles
    12-12
  • 10.
    • Our molecular understanding of gene transcription came from studies involving bacteria and bacteriophages
    • Indeed, much of our knowledge comes from studies of a single bacterium
      • E. coli , of course
    • In this section we will examine the three steps of transcription as they occur in bacteria
    12.2 TRANSCRIPTION IN BACTERIA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-14
  • 11. Promoters Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Promoters are DNA sequences that “promote” gene expression: Events at this piece of DNA are needed to initiate RNA synthesis/transcription
      • More precisely, they direct the exact location for the initiation of transcription and determine when and how frequently a gene is transcribed.
    • Promoters are typically located just upstream of the site where transcription of a gene actually begins
      • The bases in a promoter sequence are numbered in relation to the transcription start site
    12-15
  • 12. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-16 Figure 12.4 The conventional numbering system of promoters Bases preceding this are numbered in a negative direction There is no base numbered 0 Bases to the right are numbered in a positive direction Most of the promoter region is labeled with negative numbers
  • 13. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-17 Figure 12.4 The conventional numbering system of promoters The promoter may span a large region, but specific short sequence elements are particularly critical for promoter recognition and activity level Sometimes termed the Pribnow box, after its discoverer Sequence elements that play a key role in transcription
  • 14. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-18 Figure 12.5 Examples of –35 and –10 sequences within a variety of bacterial promoters The most commonly occurring bases For many bacterial genes, there is a good correlation between the rate of RNA transcription and the degree of agreement with the consensus sequences
  • 15. Initiation of Bacterial Transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • RNA polymerase is the enzyme that catalyzes the synthesis of RNA
    • In E. coli , the RNA polymerase holoenzyme is composed of
      • Core enzyme
        • Five subunits =  2  ’ 
      • Sigma factor
        • One subunit = 
      • These subunits play distinct functional roles
    12-19
  • 16. Initiation of Bacterial Transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The RNA polymerase holoenzyme binds loosely to the DNA
    • It then scans along the DNA, until it encounters a promoter region
      • When it does, the sigma factor recognizes both the –35 and –10 regions
        • A region within the sigma factor that contains a helix-turn-helix structure is involved in a tighter binding to the DNA
      • Refer to Figure 12.6
    12-20
  • 17. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-21 Figure 12.6 Amino acids within the  helices hydrogen bond with bases in the promoter sequence elements
  • 18. 12-23 Figure 12.7
  • 19. 12-26 Similar to the synthesis of DNA via DNA polymerase Figure 12.8 On average, the rate of RNA synthesis is about 43 nucleotides per second!
  • 20. Termination of Bacterial Transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Termination is the end of RNA synthesis
      • It occurs when the short RNA-DNA hybrid of the open complex is forced to separate
        • This releases the newly made RNA as well as the RNA polymerase
    • E. coli has two different mechanisms for termination
      • 1. rho-dependent termination
        • Requires a protein known as  (rho)
      • 2. rho-independent termination
        • Does not require 
    12-27
  • 21. 12-28 r ho ut ilization site Rho protein is a helicase  -dependent termination Figure 12.10
  • 22. 12-29  -dependent termination Figure 12.10
  • 23.
    •  -independent termination is facilitated by two sequences in the RNA
      • 1. A uracil-rich sequence located at the 3’ end of the RNA
      • 2. A stem-loop structure upstream of the Us
    12-30 U RNA -A DNA hydrogen bonds are very weak No protein is required to physically remove the RNA from the DNA This type of termination is also called intrinsic Stabilizes the RNA pol pausing  -independent termination Termination in Eukaryotes is much less well defined ! Figure 12.11
  • 24.
    • Many of the basic features of gene transcription are very similar in bacteria and eukaryotes
    • However, gene transcription in eukaryotes is more complex
      • Larger organisms and cells
      • Cellular complexity such as organelles
        • added complexity means more genes
      • Multicellularity: many different cell types
        • increased regulation to express only in right cells at right time
    12.3 TRANSCRIPTION IN EUKARYOTES Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-31
  • 25. Eukaryotic RNA Polymerases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Nuclear DNA is transcribed by three different RNA polymerases
      • RNA pol I
        • Transcribes all rRNA genes (except for the 5S rRNA)
      • RNA pol II
        • Transcribes all structural genes
          • Thus, synthesizes all mRNAs
        • Transcribes some snRNA genes
      • RNA pol III
        • Transcribes all tRNA genes
        • And the 5S rRNA gene
    12-32
  • 26. Sequences of Eukaryotic Structural Genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Eukaryotic promoter sequences are more variable and often much more complex than those of bacteria
    • For structural genes, at least three features are found in most promoters
      • Regulatory elements
      • TATA box (present in ~20 % of our genes) and other short sequences in TATA-promoters that have a similar function
      • Transcriptional start site
      • Refer to Figure 12.13
    12-34
  • 27.
    • The core promoter is relatively short
      • It consists of the TATA box
        • Important in determining the precise start point for transcription
    • The core promoter by itself produces a low level of transcription
      • This is termed basal transcription
    12-35 Usually an adenine Figure 12.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 28.
    • Regulatory elements affect the binding of RNA polymerase to the promoter
      • They are of two types
        • Enhancers
          • Stimulate transcription
        • Silencers
          • Inhibit transcription
      • They vary widely in their locations, from –50 to –100 region
    12-36 Figure 12.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 29. Sequences of Eukaryotic Structural Genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Factors that control gene expression can be divided into two types, based on their “location”
      • cis -acting elements
        • DNA sequences that exert their effect only over a particular gene
        • Example: TATA box, enhancers and silencers
      • trans -acting elements
        • Regulatory proteins that bind to such DNA sequences
    12-37
  • 30. RNA Polymerase II and its Transcription Factors Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Three categories of proteins are required for basal transcription to occur at the promoter
      • RNA polymerase II
      • Five different proteins called general transcription factors (GTFs)
      • A protein complex called mediator
    12-38
  • 31. 12-39 Figure 12.14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 32. 12-40 Figure 12.14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A closed complex Released after the open complex is formed RNA pol II can now proceed to the elongation stage
  • 33. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Basal transcription apparatus
      • RNA pol II + the five GTFs
    • The third component for transcription is a large protein complex termed mediator
      • It mediates interactions between RNA pol II and various regulatory transcription factors
      • Its subunit composition is complex and variable
      • Mediator appears to regulate the ability of TFIIH to phosphorylate CTD
        • Therefore it plays a pivotal role in the switch between transcriptional initiation and elongation
    12-41
  • 34. Chromatin Structure and Transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The compaction of DNA to form chromatin can be an obstacle to the transcription process
    • Most transcription occurs in interphase
      • Then, chromatin is found in 30 nm fibers that are organized into radial loop domains
        • Within the 30 nm fibers, the DNA is wound around histone octamers to form nucleosomes
    12-43
  • 35. Chromatin Structure and Transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The histone octamer is roughly five times smaller than the complex of RNA pol II and the GTFs
    • The tight wrapping of DNA within the nucleosome inhibits the function of RNA pol
    • To circumvent this problem, the chromatin structure is significantly loosened during transcription
    • Two common mechanisms alter chromatin structure
    12-44
  • 36. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • 1. Covalent modification of histones
      • Amino terminals of histones are modified in various ways
        • Acetylation; phosphorylation; methylation
    12-45 Figure 12.15 Adds acetyl groups, thereby loosening the interaction between histones and DNA Removes acetyl groups, thereby restoring a tighter interaction
  • 37. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • 2. ATP-dependent chromatin remodeling
      • The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible
    12-46 Figure 12.15 Proteins are members of the SWI/SNF family Acronyms refer to the effects on yeast when these enzyme are defective Mutants in SWI are defective in mating type swi tching Mutants in SNF are s ucrose n on- f ermenters These effects may significantly alter gene expression
  • 38. ‘ promoter’ Protein coding Difference in gene structure between - prokaryote - eukaryote core ‘ promoter’ An important difference between prokaryotes and eukaryotes is that eukaryotes’ genes are not split into intons and exons in eukaryotes is the DNA coding protein are. Therefore, exons eventually end up in the mRNA intron exons
  • 39. Pre-mRNA Transcription start, elongation, termination and RNA processing in eukaryotes : coding protein : non-coding protein: ‘leader’ and ‘trailer’ CAP CAP (poly A tail) The longest gene in human genome is more than 1.500.000 base pares (bp) and the mRNA is ~ 7000 nt. That means: >1.493.000 bp intron = ~ 99,5 % !!!!! ‘ promoter’ intron exons GENE mRNA AAAAAAAAAAAAAAn
  • 40.
    • Instead, coding sequences, called exons , are interrupted by intervening sequences or introns
    • Transcription produces the entire gene product
      • Introns are later removed or excised
      • Exons are connected together or spliced
    • This phenomenon is termed RNA splicing
      • It is a common genetic phenomenon in eukaryotes
      • Occurs occasionally in bacteria as well
    12.4 RNA MODIFICATION Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-48
  • 41.
    • Aside from splicing, RNA transcripts can be modified in several ways
      • For example
        • Trimming of rRNA and tRNA transcripts
        • 5’ Capping and 3’ polyA tailing of mRNA transcripts
    12.4 RNA MODIFICATION Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-49
  • 42. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-65 Figure 12.20
    • In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA
      • Also as heterogeneous nuclear RNA (hnRNA)
    • This RNA is altered by splicing and other modifications, before it leaves the nucleus
    • Splicing in this case requires the aid of a multicomponent structure known as the spliceosome
  • 43. Pre-mRNA Splicing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The spliceosome is a large complex that splices pre-mRNA
    • It is composed of several subunits known as snRNPs (pronounced “snurps”)
      • Each snRNP contains s mall n uclear RN A and a set of p roteins
    12-67
  • 44. Pre-mRNA Splicing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The subunits of a spliceosome carry out several functions
      • 1. Bind to an intron sequence and precisely recognize the intron-exon boundaries
      • 2. Hold the pre-mRNA in the correct configuration
      • 3. Catalyze the chemical reactions that remove introns and covalently link exons
    12-68
  • 45. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-69 Figure 12.21
    • Intron RNA is defined by particular sequences within the intron and at the intro-exon boundaries
    • The consensus sequences for the splicing of mammalian pre-mRNA are shown in Figure 12.21
    Sequences shown in bold are highly conserved Corresponds to the boxed adenine in Figure 12.22 Serve as recognition sites for the binding of the spliceosome
  • 46. 12-70 Intron loops out and exons brought closer together Figure 12.22 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 47. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-71 Figure 12.22 Intron will be degraded and the snRNPs used again
  • 48. Intron Advantage? Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • One benefit of genes with introns is a phenomenon called alternative splicing
    • A pre-mRNA with multiple introns can be spliced in different ways
      • This will generate mature mRNAs with different combinations of exons
    • This variation in splicing can occur in different cell types or during different stages of development
    12-72
  • 49. Intron Advantage? Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single gene
    • This allows an organism to carry fewer genes in its genome
    12-73
  • 50. Capping: marking 5’ends of mRNAs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’ end
      • This event is known as capping
    • Capping occurs as the pre-mRNA is being synthesized by RNA pol II
      • Usually when the transcript is only 20 to 25 bases long
    12-74
  • 51. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-75 Figure 12.23
  • 52. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-76 Figure 12.23
  • 53. Function of Capping Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • The 7-methylguanosine cap structure is recognized by cap-binding proteins
    • Cap-binding proteins play roles in the
      • Movement of some RNAs into the cytoplasm
      • Early stages of translation
      • Splicing of introns
    12-77
  • 54. The 3’ end of a mRNA: Tailing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
    • Most mature mRNAs have a string of adenine nucleotides at their 3’ ends
      • This is termed the polyA tail
    • The polyA tail is not encoded in the gene sequence
      • It is added enzymatically after the gene is completely transcribed
    12-78
  • 55. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-79 Figure 12.24 Consensus sequence in higher eukaryotes Appears to be important in the transport and stability of mRNA and the translation of the polypeptide Length varies between species From a few dozen adenines to several hundred