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Jerald D. Hendrix Last Updated: Summer 2016 Cell Architecture Review of Cell Chemistry How Cells are Studied Transcription, RNA Processing, and Transcriptional Regulation Translation and the Genetic Code Alberts, Panel 1-2 Panel 2–1 Chemical bonds and groups Panel 2–2 The chemical properties of water Panel 2–3 An outline of some of the types of sugar Panel 2–4 Fatty acids and other lipids Panel 2–5 The 20 amino acids found in proteins Panel 2–6 A survey of the nucleotides Panel 2–7 The principal types of weak noncovalent bonds Panel 3–1 Free energy and biological reactions Panel 4–1 A few examples of some general proteins Panel 4–2 Four different ways of depicting a small protein Visualization Approach Microscopy Alberts Panel 1-1 X-ray diffraction crystallography Biochemistry Approach Making Antibodies Alberts Panel 4-3 Fractionation Alberts Panel 4-4 Chromatography Alberts Panel 4-6 Electrophoresis Alberts Panel 4-7 Genetic Approach A. B. C. D. E. F. G. Structure of RNA Major Classes of RNA Transcription in Prokaryotes Transcription in Eukaryotes Post-transcriptional Processing of Eukaryotic mRNA Transcriptional Regulation in Prokaryotes: the Lac Operon as an example Transcriptional Regulation in Eukaryotes: Steroid Hormones as an Example 1. 2. 3. 4. Uracil instead of Thymine Ribose instead of Deoxyribose Usually single-stranded May have hairpin loops (e.g. loops in tRNA) Messenger RNA 1. mRNA Contains information for the amino acid sequences of proteins Transfer RNA 2. tRNA Attaches to an amino acid molecule and interfaces with mRNA during translation Ribosomal RNA 3. rRNA Structural component of ribosomes Small nuclear RNA 4. snRNA Component of small ribonucleoprotein particles Processing of mRNA Small nucleolar RNA 5. snoRNA Processing of rRNA Small cytoplasmic RNAs 6. Variable functions; many are unknown Micro RNA 7. miRNA Inhibits translation of mRNA Small interfering RNA 8. siRNA Triggers degradation of other RNA molecules Piwi-interacting RNA 9. piRNA Thought to regulate gametogenesis Requires a double-stranded DNA template 1. The DNA strands separate, and only one of the strands is used as a template for transcription “Template strand” and “nontemplate strand” Direction and numbering conventions 2. From the 3’ 5’ direction on the template strand is called “downstream” From the 5’ 3’ direction on the template strand is called “upstream” The nucleotide at the transcriptional start site is designated “+1” and the numbering continues +2, +3, etc. in the downstream direction The nucleotide immediately upstream from +1 is designated “-1” (there is no 0); numbering continues -1, -2, etc. in the upstream direction 3. 4. 5. Transcription requires nucleoside triphosphates (NTPs; ATP, GTP, CTP, UTP) as raw materials Nascent RNA strand synthesis (elongation) occurs only in the 5’ 3’ direction, with new nucleotides added to the 3’ end of the nascent strand Transcription is catalyzed by DNA-directed RNA polymerases The initiation of transcription occurs when RNA polymerase binds to a “promoter region” upstream from the transcriptional start site Promoter regions typically have short stretches of common nucleotide sequences, found in most promoters, called “consensus sequences” Common prokaryotic (bacterial) consensus sequences include: 6. 7. 8. -10 consensus sequence: TATAAT box or Pribnow box -35 consensus sequence: TTGACA -40 to -60: Upstream element; repetitive A-T pairs Bacterial RNA polymerase consists of a core enzyme and a sigma factor Bacterial RNA polymerase core has 4 or 5 subunits 9. 10. 11. α2ββ‘ω α2ββ‘ is essential; ω is not Sigma factors (σ) are global regulatory units. Most bacteria possess several different sigma factors, each of which mediate transcription from several hundred genes … … for example: 11. 12. In E. coli, during log (exponential) growth, the major sigma factor present is σ70 During stationary phase, it is σS Shifting from σ70 to σS activates the transcription of multiple genes linked to survival during stationary phase Transcription begins when the core RNA polymerase attaches to a sigma factor to form a holoenzyme molecule 13. 14. 15. 16. The holoenzyme binds to a promoter, and the dsDNA template begins to unwind A nascent RNA strand is started at +1 on the template After transcription is initiated, the sigma factor often dissociates from the holoenzyme RNA polymerase moves 3’ 5’ along the template, synthesizing the nascent RNA 5’ 3’ 17. 18. 19. Transcription ends (termination) when RNA polymerase reaches a terminator sequence, usually located several bases upstream from where transcription actually stops Some terminators require a termination factor protein called the rho factor (); these are rho-dependent. Others are rhoindependent. Messenger RNA in bacteria is often polycistronic, which means that it has the code for >1 protein on a single mRNA molecule; mRNA in eukaryotes is almost always monocistronic 1. 2. Chromatin in eukaryotes is unfolded to permit access to the template DNA during transcription Eukaryotic promoters Recognized by accessory proteins that recruit different RNA polymerases (I, II, or III) Consist of a core promoter region and a regulatory promoter region Core promoter region is immediately upstream from the coding region Usually contains: TATA box – Consensus sequence at -25 to -30 and other core consensus sequences 2. … Regulatory promoter region Immediately upstream from the core promoter, from about -40 to -150 Consensus sequences include: OCT box GC box CAAT box 3. Eukaryotic RNA polymerases RNA polymerase I: Synthesizes pre-rRNA RNA polymerase II: Synthesizes pre-mRNA RNA polymerase III: Synthesizes tRNA, 5S rRNA, and several small nuclear and cytosol RNAs Also, the different RNA polymerases use different mechanisms for termination 1. 2. In eukaryotes, mRNA is initially transcribed as precursor mRNA (“pre-mRNA”). This is part of a transcript called heterogeneous nuclear RNA (hnRNA); the terms hnRNA and pre-mRNA are sometimes used interchangably. Almost all eukaryotic genes contain introns: noncoding regions that must be removed from the pre-mRNA. The coding regions are called exons. 3. 4. 5. Introns are removed, and the exons are spliced together, by ribonucleoprotein particles called spliceosomes. mRNA contains a “leader sequence” at its 5’ end, before the coding region. The coding region begins with a translational initiation codon (AUG). A methylated guanosine cap is added to the 5’ end of the mRNA by capping enzymes. The cap is attached by a 5’ 5’ triphosphate linkage 6. 7. 8. The coding region ends with one or more translational termination codons (stop codons). At the 3’ end is a noncoding trailer region. A 3’ poly-A tail, consisting of 50 – 250 adenosine nucleotides, is added to the 3’ end by a 3’ terminal transferase enzyme. 1. 2. Operon: A group of genes in bacteria that are transcribed and regulated from a single promoter Constitutive vs. regulated gene expression Constitutive gene expression: When a gene is always transcribed Regulated gene expression: When a gene is only transcribed under certain conditions 3. The lac operon in E. coli consists of: 3 structural genes (genes that encode mRNA) lac z gene: Encodes β-galactosidase lac y gene: Encodes β-galactoside permease lac a gene: Encodes β-galactoside transacetylase The lac promoter gene: lac p The lac repressor gene: lac i (constitutively expressed and transcribed from its own promoter, different from lac p) The lac operator region: lac o (which overlaps lac p and lac z) 4. The genes of the lac operon are only transcribed in the presence of lactose (or another chemically similar inducer) In the absence of lactose, the lac repressor protein binds to lac o (lac operator) and blocks RNA polymerase from binding to the promoter (lac p) In the presence of lactose: Lactose in the cell is converted to allolactose Allolactose binds to the lac repressor protein, causing it to causing it to dissociate from the operator so RNA polymerase can reach the promoter 5. Transcription of the lac operon is stimulated by conditions of low glucose concentration When glucose levels are low: Adenylate cyclase activity is high and the concentration of cyclic AMP (cAMP) is high cAMP binds to the catabolite activator protein (CAP) The cAMP/CAP complex increases the efficiency of binding of RNA polymerase to the promoter So there is increased lac transcription … 5. When glucose levels are high: 6. Adenylate cyclase activity is lowered, so cAMP levels are low This means there is much less cAMP/CAP complex And there is decreased lac transcription So … E. coli will metabolize glucose first, then lactose when the glucose runs out 1. 2. 3. 4. Steroid hormones are secreted by endocrine gland cells and travel through the bloodstream The steroid enters the cytoplasm of target cells and binds to a cytoplasmic steroid receptor protein The steroid receptor/steroid complex enters the nucleus, where it binds to regulatory sites (typically upstream from specific promoters) Transcription from some promoters may be activated (“turned on”) while transcription from other promoters may be inhibited (“turned off”) 5. 6. Once the genes that have been activated by the steroid receptor/steroid complex (primary response or early genes) have been transcribed and translated, some of the proteins may act to regulate the expression of other genes (secondary response genes), etc. So … you may have a series of different transcriptional events over a time course with early, middle, and late events A. B. C. D. E. Amino Acid and Protein Structure Formation of Aminoacyl tRNAs Ribosome structure Stages of Translation Relationship between DNA, mRNA, and Protein Sequences 1. Amino acid structure Four different groups are attached to the central carbon atom (α-carbon) Hydrogen atom Amino group (-N+H3) Carboxylic acid group (-COO-) Side chain group (“-R”): 20 different amino acids, each with a different side chain, are encoded by codons on mRNA 2. Peptides and Proteins Peptides are formed when a covalent peptide bond (an amide bond) is formed between the carboxylic acid group of one amino acid and the amino group of another amino acid. 2. … Proteins are long peptides, over 50 amino acids long and typically much longer (in the low 100s), and typically associated with some biological function The peptide chain of a protein folds into a specific three-dimensional shape necessary for the activity of the protein. The folding of the protein, and the chemistry of the protein’s active site, are dependent on the amino acid sequence of the protein. 1. 2. 3. Amino acids are covalently attached to the 3’ end of the appropriate tRNAs. This is called the acceptor end. The anticodon is a 3-base sequence on the anticodon loop of the tRNA. It is complementary to the sequence of the codon on the mRNA. The 5’ position is referred to as the “wobble base,” meaning that it may pair up with more that one partner. The reaction is catalyzed by an aminoacyl tRNA synthase. Each tRNA has its own specific synthase enzyme. 4. This is the reaction: Amino acid + ATP + tRNA aminoacyl tRNA + AMP + PPi 5. tRNA has a distinctive 3-D structure, described as a “cloverleaf,” with hairpin loops and nonstandard bases http://en.wikipedia.org/wiki/Transfer_RNA 1. Prokaryotic ribosomes 2. Large subunit: 50 S Small subunit: 30 S Total size: 70 S Eukaryotic ribosomes Large subunit: 60 S Small subunit: 40 S Total size: 80 S http://en.wikipedia.org/wiki/Ribosome 1. Initiation a. b. c. Requires the aid of initiation factor proteins The small ribosome subunit binds to the 5’ end of mRNA. The proper orientation is believed to be established by a sequence in the leader region called the Shine-Dalgarno sequence (in prokaryotes) or similar sequences. An initiation codon (AUG) is oriented on the small ribosome subunit. AUG is the codon for the amino acid methionine. Please note: Sequences on mRNA are listed, by convention, in the 5’ 3’ direction … 1. d. e. f. A molecule of methionyl tRNA (met-tRNA) binds to the initiation codon through codon-anticodon base pairing. This step requires GTP as an energy source. The large subunit binds to the small subunit to complete the initiation complex. All initiation factors are released. Some interesting facts: In prokaryotes, the methionine on the initiating methionyl tRNA is formylated (fmet-tRNA). In eukaryotes, it is not. Not every AUG codon can be an initiation codon. Sequences in the mRNA leader seem to indicate which AUG codons are initiation codons. The initial methionine may be removed after translation (posttranslational modification), so not every protein begins with a methionine. 2. Elongation a. b. c. The ribosome/mRNA complex has two sites: the P site (to which the growing peptide chain is attached) and the A site (where the next aminoacyl tRNA binds). At the beginning of elongation, the met-tRNA occupies the P site. The A site is ready to receive the next aminoacyl tRNA. The next aminoacyl tRNA binds to the ribosome/mRNA complex at the A site. An enzyme activity in the ribosome, peptidyl transferase, forms a peptide bond between the carboxyl end of the growing peptide (on the P site) and the amino end of the next amino acid (on the A site). … 2. d. e. f. The tRNA on the P site, no longer attached to an amino acid, is released. Another enzyme activity in the ribosome, called translocase, moves the ribosome so that the peptidyl tRNA is transferred from the A site to the P site. This process requires a GTP molecule as an energy source. Now the A site is ready to accept the next aminoacyl tRNA. 3. Termination a. b. When the ribosome encounters a termination codon on the mRNA (UAA, UAG, or UGA), elongation ceases. Termination factors cause the ribosome, tRNA, and mRNA to dissociate from the nascent protein chain. Sequence Nontemplate DNA strand: 5’ ATG TTT GCT AAG GAC ATC TAA 3’ Template DNA strand: 3’ TAC AAA CGA TTC CTG TAG ATT 5’ mRNA sequence: 5’ AUG UUU GCU AAG GAC AUC UAA 3’ Amino Acid Sequence: (Amino end) Met Phe Ala Lys Asp Ile (Carboxyl end) Be certain that you can read the genetic code table. Be certain that you can read the genetic code table.