M1 - Biochemistry. Nucleic Acid Structure II/Transcription I

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1 M1 - Biochemistry Nucleic Acid Structure II/Transcription I PH Ratz, PhD (Resources: Lehninger et al., 5th ed., Chapters 8, 24 & 26) 1 Nucleic Acid Structure II/Transcription I Learning Objectives: 1. To understand the common secondary and tertiary structures of nucleic acids. 2. To be able to identify major and minor grooves. 3. To have a sense of the range of sizes of DNA molecules. 4. To define a gene. (prelude to transcription) 5. To know the characteristics of prokaryotic and eukaryotic RNA polymerases. 6. To know the general classes of RNAs. 7. To understand the differences in the activities of eukaryotic and prokaryotic promoters. 2

2 Where are we (organizationally)? Tertiary st. Secondary st. (sequence) (primary & secondary structures) (folding) Figure 1.11 Lehninger 5 th ed. 3 I. The Helical Structures of DNA & RNA: Note: One turn is the length traversed by a strand through 360 degrees of helix A-form: RNA-DNA hybrids and RNA-RNA Helix axis Blue: oxygens (ribose & Pi) Yellow: phosphates Grey: bases Watson-Crick structure is the B-form of DNA: most stable, extremely hydrated. A form B form Z form Figure 8-17 Lehninger 5 th ed. Z-form: seen especially when there are repeating sequences where C alternates with G purines flip to syn conformation, and DNA folds into the Z-form. Z-form plays a role in regulation of gene 4 expression

3 I. The Helical Structures of DNA & RNA (summary): B-form: DNA/DNA 10.5 bp/turn right-handed. A-form: DNA/RNA and RNA/RNA 11 bp/turn right-handed. Z-form: DNA/DNA having alternating purine-pyrimidine runs (eg., GCGCGCGCGCGC), 12 bp/turn left-handed purines are in syn conformation major groove is more like a convex surface. II. Range of sizes of DNA molecules: Watson-Crick structure is the B-form DNA: most stable, extremely hydrated. SV40 DNA --- 5,100 bp, double-stranded circular Mitochondrial DNA ,569 bp, double-stranded circular E. coli chromosomal DNA x 10 6 bp double-stranded circular Human chromosomal DNA x 10 7 bp (average), double-stranded linear 5 An aside: Recall that mitochondria evolved from endosymbiotic bacteria For more information See section 19.5, Lehninger, 5 th ed. Figures 1.36 Lehninger 5 th ed. 6

4 A single DNA is many times (850-fold for E. coli) longer than the structure housing it - protein coat for virus, cell for pro- & eukaryotes. So how is DNA stored in such a small volume? Viral (bacteriophage T2, left) and bacterial (E. coli, right) DNA lysed from the protein coat and cell, respectively. Figures 24.1, 24.3, 24.4 Lehninger 5 th ed. 7 DNA compaction to fit into the nucleus (see Ch 24, pp ) Secondary structure DNA wraps around the small basic proteins, histone Figures 24.11, & 24-5(a) Lehninger 5 th ed. 8

5 Gene : the fundamental unit of information in living systems. A gene is a portion of a DNA sequence that encodes a useful RNA (and most often, this useful RNA then encodes a polypeptide (protein)). Most of the human nuclear genome is non-genic (i.e., spacer ) DNA. The evolutionary maintenance of large segments of spacer DNA is an enigma - its purpose remains unknown. Only ~30% of human DNA represent genes! And only ~1.5% of human DNA - the exons, or coding segments of a gene - is translated into protein!!! The remaining gene sequences are called introns intervening nontranslated DNA sequences. Snapshot of the human genome types of sequences. Figure 24.8 Lehninger 5 th ed. 9 Exons - coding segments of a gene Introns intervening non-translated DNA sequences Note that more sequence space is devoted to introns then exons in both genes shown above. Figures 24.7 Lehninger 5 th ed. 10

6 Strand labels Genomic systems other than the eukaryotic nuclear DNA, such as mammalian mitochondrial DNA or SV40 DNA, for example, are mostly composed of contiguous gene sequences with very little spacer DNA. There is colinearity of the coding nucleotide sequence of DNA and mrna and the amino acid sequence of the polypeptide chain (i.e., functional protein). The following sections will focus on the mechanisms by which genes are recognized and transcribed into useful RNA products. Codons (nucleotide triplets complementary to the template strand that code for a specific amino acid) Non-template strand (coding strand) 11 Figures 24.2 Lehninger 5 th ed. identical in base sequence to the RNA codon Transcription: The process of RNA biosynthesis (of course, on a larger scale, this is also the 1 st step in the process of expression of information stored in genes). Mechanism requires at least a - DNA template, -NTPs (nucleoside triphosphates: ATP, GTP, CTP, UTP), and -an RNA polymerase (enzyme: to speed-up a reaction that can proceed because of energy within NTPs). colored red Nontemplate (coding) strand 5 3 E. coli RNA polymerase - In the diagram at right, note the direction of nascent RNA elongation (5 to 3 ) and the direction the template strand (colored red) is read (3 to 5 ). - These are anti-parallel activities. Figures 26.1(a) Lehninger 5 th ed. (E. Coli RNA polymerase) New NTPs added here at 3 end of RNA 12

7 A. The Primary Transcripts (newly synthesized RNA molecules) - Transcription is catalyzed by the enzyme RNA polymerase, using NTPs as substrates, incorporating NMPs and releasing PPi s as the leaving group. The DNA serves only as a template and, like RNA polymerase (the enzyme), remains unchanged in the process. 5 end of RNA transcript Reading of template is Figures 26.1(b) Lehninger 5 th ed. 13 The major classes of RNA s that are made, are: 1. Ribosomal (rrnas) (80%) - structural and functional components of ribosomes used in translation (mrna-guided protein synthesis). 2. Transfer (trnas) (17%) using an anticodon, read the information encoded in the mrna codon, and carry the amino acids to the growing polypeptide chain on the ribosome in translation. 3. Messenger (mrnas) (2%) encode the primary amino acid sequence of one or more polypeptides specified by a gene or gene-set based on the linear array of codons. 4. Other RNAs (1%) - Includes such minor species as small nuclear RNAs and ribozymes. 14

8 B. The RNA Polymerases: 1. Prokaryotes - Have one version of RNA polymerase that synthesizes all classes of RNAs. This enzyme is guided by a loosely bound subunit called σ that has special affinity for DNA promoter sequences. The common σ is called σ70, which has specificity for the constitutive genes. The diagram below shows the subunit structure of Thermus acquaticus RNA polymerase very similar to E. coli RNA polymerase: (α 2 ββ ω and σ) The active site for transcription is in the cleft between β & β Figures 26.4 Lehninger 5 th ed. 15 RNA polymerase holoenzyme: Necessary for initiation of transcription Prokaryotic RNA polymerase shown with σ bound is referred to as the holoenzyme. Without σ, it is called the core enzyme. When σ is bound, the enzyme has higher affinity for promoter sequences and lesser affinity for nonpromoter sequences. Once the initiation of transcription is complete, σ dissociates from the holoenzyme and binds another core enzyme. Core enzyme Holoenzyme To another core E Figures 26.6(a) part 2. Lehninger 5 th ed. 16

9 rrna (-5S rrna) mrna (+snrna) trna (+5S rrna) 2. Eukaryotes -Have three different RNA polymerases that function with (are recruited to) three different types of promoters (different promotor sequences): a. RNA Polymerase I (Pol I) - Recognizes rrna genes, except 5S rrna. Is responsible for the synthesis of only one type of RNA a transcript called pre-ribosomal RNA (pre-rrna). b. RNA Polymerase II (Pol II) - Recognizes mrna genes and most snurp RNA genes (a specialized RNA). Recognizes thousands of promoters that vary greatly in sequence. c. RNA Polymerase III (Pol III) - Recognizes small RNA genes such as the trna and 5S rrna genes as well as some other small specialized RNAs (stable RNAs like U6 snrna). d. Eukaryotes also have a mitochondrial RNA Polymerase that synthesizes all mitochondrial RNAs. Division of labor 17 Bottom line in comparing prokaryotic and eukaryotic RNA polymerase 3D structures: they look almost identical and have the same evolutionary origin. All eukaryotic RNA polymerases are homologous to one another and to prokaryotic RNA polymerase - but eukaryotic RNA polymerases display additional complexities (additional subunits, etc.) 18

10 Promoters: Specific DNA sequences where RNA synthesis begins. The gene numbering system - The transcription start site is designated +1 - The downstream (direction of transcription) base pairs are counted +2, +3, +4,. - The base pairs upstream of +1 are designated -1, -2, -3, -4,. - There is no zero. DNA, -4, -3, -2, -1 +1, +2, +3, +4, The gene to be transcribed Why are upstream sequences important? 19 The prokaryotic promoter - Has two major regions just upstream of the transcription start site that are recognized by the holoenzyme. The TATA box around -10 and the sequence around -35. Their respective consensus sequences, and the less common AT-rich UP (upstream) element, are diagrammed below, along with some specific examples. Be aware that these promoter elements are double stranded, but they are designated (by convention) by their non-template 5 to 3 strand sequences. 5 3 The UP element is bound by Pol α subunit Figures 26.5 Typical E. coli promoters for 5 different genes recognized by σ 70 of 20 RNA Pol holoenzyme. Lehninger 5 th ed.

11 Transcription initiation and elongation in E. coli. The promoter can loosely be defined as the sum of DNA sequences necessary for transcription initiation. ~10 RNA nucleotides Figures 26.6 Lehninger 5 th ed. 21 So, if the unique feature of DNA is on the inside of the helix, how do RNA Polymerases (and gene regulatory proteins) recognize discrete sequences? Different base pairs can be recognized from their edges when viewed at the major, but not minor, groove. Note that gene regulatory proteins usually bind to the major groove. Figures 7.7 & 7.8, Lodish, Molec Biol of the Cell, 4 th ed. 22

12 The eukaryotic promoter DNA sequences are diverse in that the specific recognition elements are different for the different polymerase classes and even within a class. Perhaps the most diverse are the Pol II promoters for which it is difficult to deduce a consensus. One of many possible examples is shown below. Note that although the TATA box and initiator sequence (Inr) shown here is a Pol II promotor consensus sequence for most eukaryote genes, some Pol II promotors lack the TATA box, Inr, or both. Figures 26.9 Common sequences in many promotors recognized by eukaryotic RNA Pol II. Lehninger 5 th ed. 23 In addition to RNA Pol II, other proteins are required for transcription initiation & elongation in eukaryotes: DNA sequences in eukaryotic promoters are recognized by proteins other than the RNA polymerase: TRANSCRIPTION FACTORS these guide RNA Pol II to the start site. 2 (TFII? = Transcription Factor for Pol II ) TBP is a component of TFIID CTD = carboxy-terminal domain of Pol II 24

13 Assembly of RNA Pol II & transcription factors at a promoter Carboxy-terminal domain (CTD) plays important roles in Pol II function. Note that phosphorylation of this region permits escape from the promoter & activates the beginning of transcription (i.e., elongation) Assembly of general TFs on promotor sequence Figures 26.10(a) Transcription at RNA Pol II promotors. Lehninger 5 th ed. 25 Eukaryotic transcription factors - Typically there are about a dozen transcription factors (proteins), besides the RNA polymerase II, that are required for initiation and elongation on a gene. I suppose transcription factors (TFs) could be considered the eukaryotic counterparts of σ, however, rather than binding the polymerase itself, they act differently in that most bind cis elements (i.e., regulatory DNA sequences on the same molecule of DNA as the transcribed DNA sequence that is being regulated) in the promoter, and establish a high affinity landing port for the proper polymerase. Fig Lodish, Molec Cell Biol, 5 th ed. Phosphorylation of the CTD Fig 6-18, Alberts, 4 th ed. DNA bending induced by TBP binding TATA region 26

14 Activation of transcription initiation in eukaryotes by recruitment of RNA Pol II holoenzyme. Note the cis gene regulatory element bound by an transcriptional activator protein that attracts the holoenzyme complex to the promotor (and helps the RNA Pol II and general TFs in overcoming the difficulty of binding to DNA packaged in chromatin). You ll hear more about this in later lectures. DNA bound activator proteins typically activate the rate of transcription by up to 1000-fold Figures 7.43, Lodish, Molec Biol of the Cell, 4 th ed. 27