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Principles of Biochemistry

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1 Principles of Biochemistry
Horton • Moran • Scrimgeour • Perry • Rawn Principles of Biochemistry Fourth Edition Chapter 21 Transcription and RNA Processing Copyright © 2006 Pearson Prentice Hall, Inc.

2 Chapter 21 - Transcription and RNA Processing
Gene - a DNA sequence that is transcribed (includes genes that do not encode proteins) “Housekeeping genes” encode proteins or RNA essential for normal activities of the cell (e.g. enzymes in basic metabolic pathways, tRNAs and rRNAs) Biological information flow from DNA to proteins is irreversible

3 Fig 21.1 Biological information flow

4 21.1 Types of RNA (1) Transfer RNA (tRNA)
Carries amino acids to translation machinery Very stable molecules (2) Ribosomal RNA (rRNA) Makes up much of the ribosome Very stable, majority of cellular RNA (3) Messenger RNA (mRNA) Encodes message from DNA to ribosomes Rapidly degraded by nucleases

5 Table 21.1

6 21.2 RNA Polymerase RNA polymerase (RNA pol) catalyzes DNA-directed RNA synthesis (transcription) RNA pol is core of a larger transcription complex Complex assembles at one end of a gene when transcription is initiated DNA is continuously unwound as RNA pol catalyzes a processive elongation of RNA chain

7 A. RNA Polymerase Is an Oligomeric Protein
Core enzyme: a2bb’w participates in many of the transcription reactions b Subunit contains part of polymerase site b’ Subunit contributes to DNA binding a Subunits are scaffolding and interact with other proteins that regulate transcription w Role not well characterized s Subunit role in initiation of transcription

8 Table 21.2

9 Fig 21.2 Structure of T. aquaticus RNA pol core enzyme

10 B. The Chain Elongation Reaction
Mechanism almost identical to that for DNA polymerase Growing RNA chain is base-paired to DNA template strand Incoming ribonucleotide triphosphates (RTPs) form correct H bonds to template New phosphodiester bond formed, PPi released

11 RNA polymerase reaction
Catalyzes polymerization in 5’ 3’ direction Is highly processive, and thermodynamically assisted by PPi hydrolysis Incoming RTPs: UTP, GTP, ATP, CTP Growing single-stranded RNA released Adds nucleotides/sec (~ 1/10th rate of DNA replication)

12 Fig 21.3 RNA polymerase reaction

13 Fig 21.3 RNA polymerase reaction

14 Fig 21.3 (cont)

15 21.3 Transcription Initiation
Transcription complex assembles at an initiation site (DNA promoter region) Short stretch of RNA is synthesized Operon: a transcription unit in which several genes are often cotranscribed in prokaryotes Eukaryotic genes each have their own promoter

16 Fig 21.4 Transcription of E. coli ribosomal RNA genes

17 A. Genes Have a 5’ → 3’ Orientation
Convention for double-stranded DNA: Coding strand (top) is written: 5’ → 3’ Template strand (bottom) is written: 3’ → 5’ Gene is transcribed from 5’ end to the 3’ end Template strand of DNA is copied from the 3’ end to the 5’ end Growth of RNA chain proceeds 5’ → 3’

18 Fig 21.5 Orientation of a gene

19 B. Transcription Complex Assembles at a Promoter
Consensus sequences are found upstream from transcription start sites DNA-binding proteins bind to promoter sequences (prokaryotes and eukaryotes) and direct RNA pol to the promoter site The s subunit of prokaryotic RNA polymerases is required for promoter recognition and formation of the complex

20 Fig 21.6 Promoter sequences from 10 bacteriophage and bacterial genes

21 E. coli Promoter (1) TATA box (-10 bp upstream from transcription start site (rich in A/T bp) (2) -35 region (-35 bp upstream) from start site A s subunit also required (e.g. s70) Strong promoters match consensus sequence closely (operons transcribed efficiently) Weak promoters match consensus sequences poorly (operons transcribed infrequently)

22 C. The s Subunit Recognizes the Promoter
s Subunit increases the affinity of the core polymerase for specific promoter sequences s Subunit also decreases the affinity of the core polymerase for nonpromoter regions Core polymerase (no s subunit) binds DNA nonspecifically

23 Table 21.3

24 D. RNA Polymerase Changes Conformation
Unwinding of DNA at the initiation site requires a conformational change RNA pol (R) and promoter (P) shift from: (RPc) closed complex (DNA double stranded) (RPo) open complex (18 bp DNA unwound) (forms transcription bubble)

25 Fig 21.7 Initiation of transcription in E. coli

26 Fig 21.7 Initiation of transcription in E. coli (two slides)

27 Fig 21.7 (continued)

28 Animations Transcription

29 21.4 Transcription Termination
Only certain regions of DNA are transcribed Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences Two types of termination sequences: (1) Unstable elongation complex (2) Rho-dependent termination

30 Pause Sites Pause sites - regions of the gene where the rate of elongation slows down (10 to 100-fold) or stops temporarily Transcription termination often occurs here G-C- rich regions are more difficult to separate than A-T rich regions and may be pause sites Pause is exaggerated when newly transcribed RNA can form a hairpin

31 Fig 21.8 Formation of an RNA hairpin

32 Rho-dependent Termination Sites
Rho triggers disassembly of the transcription complex at some pause sites Rho binds to ssRNA chain, destabilizing the RNA-DNA hybrid and terminating transcription

33 Fig 21.9 Rho-dependent termination of transcription (E. coli)
RNA pol is stalled at pause site Rho binds to new RNA, destabilizes RNA-DNA hybrid

34 21.5 Transcription in Eukaryotes
A. Eukaryotic RNA Polymerases Three different RNA polymerases transcribe nuclear genes Other RNA polymerases found in mitochondria and chloroplasts Table 21.4 (next slide) summarizes these RNA polymerases


36 Fig 21.10 RNA polymerase II from S. cerevisiae (yeast)

37 Fig 21.11 A Generic Eukaryotic Promoter

38 B. Eukaryotic Transcription Factors
Same reactions as prokaryotic transcription More complicated assembly of machinery Binding of RNA polymerase to promoters requires a number of initiation transcription factors (TFs)

39 Table 21.5

40 RNA polymerase II Transcribes all protein-encoding genes and some small RNA encoding genes Protein-encoding RNA synthesized by RNA pol II is called mRNA precursor (or hnRNA) 20%-40% of all cellular RNA synthesis General transcription factors interact directly with RNA pol II and control initiation at class II genes

41 Fig 21.12 A. thaliana TATA-binding protein (TBP) (blue) bound to DNA

42 Fig 21.13 RNA polymerase II holoenzyme complex bound to a promoter

43 B. The Role of Chromatin in Eukaryotic Transcription
Status of chromatin affects gene expression Chromatin status is modulated by Post-transcriptional modification of histones, e.g. methlylation or acetylation

44 21.6 Transcription of Genes Is Regulated
Expression of housekeeping genes is constitutive (constitutive genes) These genes usually have strong promoters and are efficiently and continuously transcribed Housekeeping genes whose products are required at low levels have weak promoters and are infrequently transcribed Regulated genes are expressed at different levels under different conditions

45 Role of regulatory proteins in transcription initiation
Regulatory proteins bind to specific DNA sequences and control initiation of transcription Repressors - regulatory proteins that prevent transcription of a negatively regulated gene Negatively regulated genes can only be transcribed in the absence of the repressor Activators - regulatory proteins that activate transcription of a positively regulated gene

46 Inducers and Corepressors
Repressors and activators are often allosteric proteins modified by ligand binding Inducers - ligands that bind to and inactivate repressors Corepressors - ligands that bind to and activate repressors Four general strategies for regulating transcription (Figure 21.14, next four slides)

47 Fig Strategies for regulating transcription initiation by regulatory proteins




51 Animations Transcriptional Regulation

52 21.7 The lac Operon, an Example of Negative and Positive Regulation
E. coli can use lactose and other b-galactosides as a carbon source Uptake and catabolism of b-galactosides requires three proteins When glucose is present these proteins are synthesized in limited amounts When glucose levels are low these proteins are synthesized in larger amounts

53 The Proteins of E. coli b-Galactoside Metabolism
(1) Lactose permease (lacY gene product) - transporter for uptake of b-galactosides (2) b-Galactosidase (lacZ gene product) - hydrolyzes b-galactosides to hexoses (3) Thiogalactoside transacetylase (lacA gene product) - acetylates (add CH3CO-) nonmetabolizable b-galactosides prior to elimination from the cell

54 The lac Operon Three genes - lacZ, lacY and lacA form an operon that is transcribed from a single promoter Resulting large mRNA molecule contains three separate protein-coding regions (3 genes) Operons with multiple protein-coding regions are common in prokaryotes but not eukaryotes

55 A. lac Repressor Blocks Transcription
lac Operon expression is controlled by the lac repressor protein lac Repressor is encoded by the lacI gene, located upstream from the lac operon lac Repressor binds simultaneously to 2 repressor-binding sites (operators) Operator (O1) is adjacent to the promoter, (O2) is within the coding region of lacZ

56 Fig 21.15 Organization of the genes for proteins required to metabolize lactose
Three coding regions of lac operon are cotranscribed from the Plac promoter lacI is under the control of the P promoter

57 Repressor-operator Interactions
The lac repressor binds simultaneously to both operators O1 and O2 This causes the DNA to form a stable loop which blocks initiation of transcription b-Galactosides act as inducers to cause the repressor to dissociate from the operon allowing transcription to continue

58 Fig 21.16 Electron micrographs of DNA loops

59 Fig 21.17 Binding of lac repressor to the lac operon
Tetrameric lac repressor interacts simultaneously with two sites near the lac promoter DNA loop forms RNA pol can still bind to the promoter

60 Allolactose Inducer for the lac Operon
In the absence of lactose, the lac repressor blocks expression of the lac operon Several b-galactosides can act as inducers to start gene transcription In the presence of lactose, allolactose is the inducer b-Galactosidase converts lactose to allolactose

61 Fig 21.18 Formation of allolactose from lactose

62 B. The Structure of the lac Repressor
Left: End-on view Right: Side view, lac repressor a-helix in the major groove

63 C. cAMP Regulatory Protein Activates Transcription
The lac operon is transcribed maximally when b-galactosides are the only carbon source When glucose is present transcription is reduced 50-fold, termed catabolite repression A weak promoter in these operons is promoted to a stronger one by an activator (in the absence of glucose)

64 Activation of the lac Operon
cAMP regulatory (or receptor) protein (CRP) is the activator CRP is also known as catabolite activator protein (CAP) In the presence of cAMP, CRP binds near the promoters of more than 30 genes RNA polymerase is activated and the rate of transcription activation is increased

65 Fig 21.20 Activation of transcription initiation at the lac promoter by CRP-cAMP

66 Fig (cont)

67 Fig cAMP production In the absence of glucose, enzyme III (EIII) transfers a phosphate group to adenylate cyclase leading to CRP-cAMP increases CRP-cAMP activates transcription of other genes

68 Fig 21.22 Conformational changes in CRP caused by cAMP binding
a-Helices of each monomer of the cAMP-CRP dimer fit into major groove of DNA

69 Fig 21.23 Structure of CRP-cAMP and DNA complex
Both subunits have a cAMP bound at the allosteric site Each subunit has an a-helix in DNA major groove

70 21.8 Posttranscriptional Modification of RNA
RNA processing Removal of nucleotides from primary RNA transcripts Addition of nucleotides with sequences not encoded by corresponding genes Covalent modification of certain bases

71 21.8 Posttranscriptional Modification of RNA
Mature tRNA molecules are generated in both prokaryotes and eukaryotes by processing the primary transcripts, termed RNA processing. In prokaryotes, the primary transcripts often contain several tRNA precursors Ribonucleases (RNases) cleave the large primary transcripts to their mature lengths

72 Fig 21.24 Summary of prokaryotic tRNA processing (three slides)



75 Fig 21.25 Examples of covalent modification of nucleotides in tRNA molecules

76 Fig 21.25 Examples of covalent modification of nucleotides in tRNA molecules

77 Fig (cont)

78 Fig (cont)

79 B. Ribosomal RNA Processing
Ribosomal RNA in all organisms are produced as large primary transcripts that require processing Processing includes methylation and cleavage by endonucleases Prokaryotic rRNA primary transcripts ~30S Contain one copy each: 16S, 23S, 5S rRNA

80 Fig 21.26 Endonucleolytic cleavage of rRNA precursors in E. coli

81 21.9 Eukaryotic mRNA Processing
In prokaryotes the primary mRNA transcript is translated directly In eukaryotes transcription occurs in the nucleus, translation in the cytoplasm Eukaryotic mRNA is processed in the nucleus without interfering with translation In some mRNA, pieces are removed from the middle and the ends joined (splicing)

82 A. Eukaryotic mRNA Molecules Have Modified Ends
All eukaryotic mRNA precursors undergo modifications to increase their stability and make them better substrates for translation Ends are modified so they are no longer susceptible to exonuclease degradation The 5’ ends are modified (formation of a cap) before the mRNA precursors are completely synthesized

83 Fig 21.27 Formation of a cap at the 5’ end of a eukaryotic mRNA precursor phosphohydrolase

84 Fig 21.27 phosphohydrolase

85 Fig (cont)

86 Fig (cont) Guanylate base is methylated at N-7 methylase

87 2- Hydroxyl groups of last two riboses may also be methylated
Fig (cont) methylase

88 Poly A tails at the 3’ ends of mRNA precursors
Eukaryotic mRNA precursors are also modified at their 3’ ends (formation of a poly A tail) A poly A polymerase adds up to 250 adenylate residues to the 3’ end of the mRNA precursor This poly A tail is progressively shortened by 3’ exonucleases The poly A tail increases the time required for nucleases to reach the coding region

89 Fig 21.28 Polyadenylation of a eukaryotic mRNA precursor

90 Fig (cont) CPSF: a cleavage and polyadenylation specificity factor

91 Fig (cont)

92 B. Some Eukaryotic mRNA Precursors are Spliced
Introns - internal sequences that are removed from the primary RNA transcript Exons - sequences that are present in the primary transcript and the mature mRNA Splice sites - junctions of the introns and exons where mRNA precursor is cut and joined

93 Fig 21.29 Triose phosphate isomerase gene (nine exons and eight introns)

94 Fig (cont) Protein 3-dimensional structure showing parts of protein encoded by each exon

95 Fig 21.30 Consensus sequences at splice sites in vertebrates
Y: pyrimidine (U or C) R: purine (A or G) N: any nucleotide

96 The Spliceosome The spliceosome is a large RNA-protein complex which catalyzes splicing reactions It contains 45 proteins and 5 small nuclear RNA (snRNA) molecules (“snurps”)

97 Fig 21.31 Intron removal in mRNA precursors

98 Fig (cont)

99 Fig (cont)

100 Fig 21.32 Formation of a spliceosome
** Each snRNP contains one or two snRNA plus a number of proteins.

101 Fig (cont)

102 Fig (cont)

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