Lect 6-Tues-Sept24-2024-Gene Transcrip and Gene Translation PDF

Course HSS 2305 A Molecular Mechanism of Disease Lecture-6 Overview: Genes, Transcription and Translation Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of Medicine, U Ottawa,...

Course HSS 2305 A Molecular Mechanism of Disease Lecture-6 Overview: Genes, Transcription and Translation Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of Medicine, U Ottawa, Roger Guindon Building 451 Smyth Road Ottawa, ON K1H 8M5 Tel 613-878-7043 (Cell) E-mail: [email protected] Alternate: [email protected] Affiliate Investigator, Chronic Disease Program, Ottawa Hospital Research Institute Web: https://med.uottawa.ca/pathology/people/basak-ajoy 1 2  CHAPTER 11 Gene Expression: From Transcription to Translation 3  Keys  Define the relationship between genes and polypeptides.  Describe the flow of information through cells.  Elaborate on differences between transcription in prokaryotes and eukaryotes.  Outline rRNA and tRNA processing to their mature forms.  Define heterogeneous nuclear RNAs and mature mRNA.  Describe the structure of mRNA, the 5' cap and the poly(A) tail.  Summarize the proposed importance of the split gene.  Summarize the processing of hnRNA to mature mRNA, emphasizing the removal of introns and splicing together of exons.  Describe ribozome involvement in RNA processing and protein synthesis.  Describe the properties of the genetic code, its theoretical underpinnings and the codon assignments.  Define the role of tRNAs in decoding the genetic code.  Summarize the steps in all stages of translation: tRNA charging, initiation, elongation and termination. 4 FROM GENES TO PROTEINS http://youtube/erOP76_qLWA m 5 FROM GENES TO PROTEINS 66 From Genes to Proteins 7 http://youtube/erOP76_qLWA  Relationship between Genes & Proteins  Genes store information for producing all cellular proteins.  Early observation suggested a direct relationship between genes and proteins.  A. Garrod studied the relationship between a specific gene, a specific enzyme, and a metabolic condition (Alcaptonuria).  G. Beadle and E. Tatum formulated the “one gene–one enzyme” hypothesis. 8 The Relationship Between Genes and Proteins It was first demonstrated by Scottish Physician A Garrod (1908) who noted a rare inherited disease called Alcaptonuria where urine turns dark upon exposure to air due to lack of an enzyme in their blood that oxidized Homogentisic acid (HA) (a compound formed during breakdown of Phe and Tyr). As HA also called Melanic acid accumulated and excreted in the urine it turned dark due to oxidation by air. Garrod had discovered the relationship between a genetic defect, a specific enzyme, and a specific metabolic condition. He called such diseases “Inborn Errors of HA (Homogentisic acid), Metabolism.” This finding remains Melanic acid, unnoticed for decades Chemical Name: 2,5-Dihydroxy- phenylacetic acid 9 Gene Directs The Production of Enzyme Experiment with Neurospora (Bread mold) 1940 Beadle and Tatum (Caltech, USA): Irradiated 1000 cells of Neurospora. Two of the cells (due to mutation) was found unable to grow in minimal medium that lacked the essential compounds known to be synthesized by the organism. They found a genetic mutant in Neurospora that grows in minimal medium only when it is supplemented with a particular coenzyme (Pantothenic acid of coenzyme-A). 10 Gene Directs The Production of Enzyme 11  Relationship Between Genes & Proteins Beadle and Tatum’s hypothesis was later modified to “one gene-one polypeptide chain” Mutation in a single gene causes a single substitution in an amino acid sequence of a single protein. If a spore can’t grow in minimal medium but can with supplemented Pantothenic acid, it concludes an enzymatic deficiency that prevents pantothenic acid production 12  Relationship Between Genes & Proteins Flow of Information An Overview of the Flow of Information through the Cell – Messenger RNA (mRNA) is an intermediate between a gene and a polypeptide. – Transcription is the process by which RNA is synthesised from a DNA template in the nucleus – Translation is the process by which proteins are synthesized in the cytoplasm using information encoded by mRNA template. Overview of the flow of information in eukaryotes 13  Mechanism By Which Specific Polypeptide Chain Is Generated Transcription Translation Gene (DNA) mRNA Protein The momentous discovery of mRNA was made in 1961 by François Jacob and Jacques Monod of the Pasteur Institute in Paris, Sydney Brenner of the University of Cambridge and Matthew Meselson of the California Institute of Technology. A messenger RNA is assembled as a complementary copy of one of the two DNA strands that make up a gene. The synthesis of an RNA from a DNA template is called TRANSCRIPTION. Because its nucleotide sequence is complementary to that of the gene from which it is transcribed, the mRNA retains the same information as the 14 gene itself. 14 Gene (A coding segment of DNA present in Chromosome) - Unwinding of double helix DNA Transcription Required machinery: - RNA Polymerase (Enzyme) - Transcription factors mRNA (messenger RNA) (End product) Required machinery: Translation - Ribosomal RNA (rRNA) - Transfer RNA (tRNA) - Ribosome (Enzyme) Polypeptide/Protein (End product) 15  The three roles of RNA in protein synthesis Messenger RNA (mRNA) is translated into protein by the joint action of transfer RNA (tRNA) & ribosome, which is composed of numerous proteins & two major ribosomal RNA (rRNA) molecules. 16 Relationship between Genes & Proteins: Classes of RNA  There are three classes of RNA in a cell: mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA).  rRNA recognizes other molecules, provide structural support, and help ribosome to catalyze the chemical reaction in which amino acids are linked to one another.  tRNAs are required to translate information in the mRNA code into 2-Dimensional structure of a amino acids. Bacterial ribosomal RNA - Extensive base-pairing between different regions of the single strand. - The expanded section shows the base sequence of a stem and loop, including a nonstandard base pair (G-U) and a modified nucleotide, methyl-Adenosine (mA). One of the helices is shaded differently because it plays an important role in ribosome. 17 An Overview of Transcription & Translation in Prokaryotic and Eukaryotic Cells 18 Transcription & Translation in Prokaryotic Cells 19  Transcription in Bacteria (Prokaryotic Cell) - Bacteria (E. coli) contains a single RNA polymerase composed of 5 subunits that form a core enzyme. If this enzyme from bacterial cells is added to a solution of bacterial DNA molecules and ribonucleoside triphosphates, the enzyme binds to the DNA and synthesizes RNA. - The RNA molecules thus produced are not the same as those found within cells since the enzyme is attached to random sites in the DNA, sites that it would normally have ignored in vivo. - If, however, a purified accessory polypeptide called “Sigma Factor” (s) is added to the RNA polymerase before it attaches to DNA, transcription begins at selected locations. Attachment of s factor increases the enzyme’s affinity for promoter sites in DNA. 20 20  The elements of a promoter region in the DNA of the E. coli Consensus Consensus sequence sequence (Pribnow box) Pribnow box  The regulatory sequences required for initiation of transcription are located at -35 & -10 base pairs from the site at which transcription begins. The initiation site marks the boundary between + and - sides of the gene. Bacterial Promoters are located in the region of a DNA strand just preceding the initiation site of RNA synthesis. Those portions of the DNA preceding the initiation site (toward the 3’ end of the template) are said to be “Upstream” from that site. Those portions of the DNA succeeding it (toward the 5’ end of the template) are said to be “Downstream” from that site. Two consensus sequences “TTGACA” and “TATAAT” (Pribnow box) are essential part of a promoter site on DNA for transcription. Sigma Factor” binds to the latter “Promoters are the sites in DNA that bind RNA polymerase” 21 Transcription & Translation in Eukaryotic Cells 22 Transcription & RNA Processing in Eukaryotic Cells RNA Polymerase: (Discovered in 1969 by Robert Roeder, U Washington) It binds to DNA and incorporates nucleoti[des into a strand of RNA whose sequence is complementary to one of the DNA strands (template). Eukaryotic cells have 3 distinct transcribing enzymes in their cell nuclei. Each of these enzymes is responsible for synthesizing a different group of RNAs 23  The Machinery for Transcription in Eukaryotic cells  All eukaryotic mRNA precursors are synthesized by RNA polymerase II, an enzyme composed of a dozen different subunits that is remarkably conserved from yeast to mammals.  RNA polymerase II binds the promoter region with the help of a number of General Transcription Factors (GTFs) to form a Pre- Initiation Complex (PIC). 24 A comparison of prokaryotic and eukaryotic RNA polymerase structure RNA polymerase II (RNAPII) one of 3 major eukaryotic nuclear RNA polymerases (Within Red Box). The surface structures of RNA polymerases from each of the three domains of life: Bacteria, Archaea and and Eukaryotes 25  Transcription: RNA Polymerase  Promoter region = Site on DNA to which RNA polymerase binds prior to initiation of transcription, determines which strand is template (anti-sense)  Large and variable, 100 - 1000 base pairs (bp) long  Core promoter elements: - TATA element (Consensus sequence, TATAAA) Recognized by transcription factor TATA-binding protein (TBP) - B recognition element (BRE) - Recognized by transcription factor TFIIB - Initiator element (INR) - Recognized by TBP-Associated Factors (TAF) 1, 2 - Downstream promoter element (DPE) - Recognized by TBP-Associated Factors (TAF) 1, 2 26 Transcription: RNA Polymerase 27 27 Transcription: Pre-Initiation Complex Pre-Initiation Complex (PIC): Helps position RNA polymerase II over gene transcription start sites Denatures the DNA Positions the DNA in the RNA polymerase II active site for transcription 28 Transcription: Pre-initiation Complex 1) TFIID (TBP + TAFs) binds to TATA box 2) Binding of TFIIA and TFIIB to complex. TFIIB provides specific binding site for RNA Pol II 29 Transcription: Pre-initiation Complex 3) The RNA Pol II – TFIIF complex binds to TFIIB in the PIC 4) TFIIE and TFIIH bind to the PIC TFIIH contains 3 enzymatic subunits Kinase Phosphorylates RNA Pol II Helicases Unwind DNA at promoter start site Hydrolysis of ATP by TFIIH can form the transcription bubble (13 bp) open formation 30 Transcription: Pre-initiation Complex Transcription initiated by phosphorylation of Carboxyl-terminal domain (CTD) of RNA Pol-II 7 amino acid (aa) repeating domain of RNA Pol II subunit (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) becomes phosphorylated by TFIIH Ser-5 triggers uncoupling of RNA Pol II from PIC and promotes elongation TFIID remains bound to TATA and can initiate formation of additional PIC 31 Transcription : Elongation Transcription Bubble: unwound section of DNA of approximately 13 bp regions DNA in front of RNA Pol are unwound, compensatory positive supercoils (Chapter 10) DNA behind RNA Pol are rewound and negative supercoils are present (Chapter 10) DNA-RNA hybrid: ~8-9 bp, stabilizes the elongation complex 32 Transcription: Elongation Complementary nucleotides are incorporated by RNA Pol II in a 5’ 3’ direction Incoming Adenosine-triphosphate pairs with the Thymine containing nucleotide of the template (H-bonds) 3`OH of the previous nucleotide sugar binds to the 5’ α-phosphate of incoming nucleoside triphosphate Pyrophosphate (PPi) is released and hydrolyzed to 2 inorganic phosphates (Pi). This releases a large amount of energy irreversible 33  Transcription: Elongation Elongation Transcription Factors: > 50 components involved in elongation P-TEFb: phosphorylates the CTD at Ser-2 leading to productive elongation and required RNA modifications TFIIF: weakens interactions between RNA Pol II and nonspecific binding sites of DNA, suppressing transient pausing of the polymerase TFIIS: stimulates elongation, RNA Pol II moving after pauses, proofread & correction of mistaken nucleotides 34 Transcription: Termination In bacterial cells: well defined termination sequences In eukaryotic cells: No well-defined sequence, not well understood 3’ end of mRNA determined by series of processing steps Cleavage of the new transcript followed by template-independent addition of Adenine (A) at its new 3' end Polyadenylation 35 Difference between DNA and RNA in chemical structure DNA (2’-Deoxyribo-Nucleic Acid) * The sugar and base alone are called a Nucleoside. * Adding a phosphate (or Phosphoester bond more than one phosphate) to a nucleoside creates a Nucleotide. Glycosidic bond Formation of Nucleotide by Removal of Water. The carbon atoms in 2’ deoxyribose are labeled in Red. dAMP: 36 Deoxy Adenosine MonoPhosphate  DNA STRUCTURE Amino - Imino and Keto - Enol tautomerism. (a) Cytosine is usually in the amino form but rarely forms the imino configuration. (b) Guanine is usually in the keto form but is rarely found in the enol configuration. 37 Structure of double helix DNA 38 DNA IS RIGHT HANDED HELIX 39  RNA STRUCTURE  RNA Contains Ribose and Uracil and is Usually Single- Stranded unlike DNA The figure shows the structure of the backbone of RNA, composed of alternating phosphate and ribose moieties. 40   G:U Base Pair. The structure shows hydrogen bonds that allow base pairing to occur between Guanine and Uracil  RNA Chains Fold Back on Themselves to Form Local Regions of Double Helix Similar to A Form of DNA Double Helical Characteristics of RNA. In an RNA molecule having regions of complementary sequences, the intervening (non-complementary) stretches of RNA may become “looped out” to form one of the structures illustrated in the figure. (a) Hairpin (b) Bulge (c) 41 Loop  TRANSLATION  It requires the participation of dozens of various components including the “Ribosomes” which are nonspecific components of the translation machinery. These complex, cytoplasmic “machines” can be programmed, to translate the information encoded by any mRNA. Ribosomes contain both protein and RNA. The RNAs of a ribosome are called ribosomal RNAs (rRNAs), and like mRNAs, each is transcribed from one of the DNA strands of a gene.  Transfer RNAs (tRNAs) constitute a third major class of RNA that is required to translate the information in the mRNA nucleotide code into the amino acid “Alphabet” of a polypeptide.  Many RNAs fold into a complex three-dimensional shape, which is markedly different from one type of RNA to another. 42 - Until recently, the way in which genes control cells was summed up by a simple formula: DNA makes RNA which makes protein, and proteins are the cellular workhorses that carry out all the crucial tasks. -“Small RNAs” do not code for proteins, but instead exercise control over those RNAs that do. -They provide a further clue to understanding the mysterious 98% of the human genome that doesn't direct the production of proteins. Eukaryotic cells make a host of other RNAs (small) (besides m, r and t-RNA) which also play vital roles in cellular metabolism. - Small nuclear (sn) - Small nucleolar (sno) - Small (short) interfering (si) - Micro (mi) 43 MicroRNAs (miRNAs) These are 22-24 nucleotides in length, and down- regulate gene expression by attaching themselves to messenger RNAs (mRNAs) and preventing them from being translated into proteins. Short interfering RNAs (siRNAs) These RNAs are around 22 nucleotides in length and mediate the recently discovered phenomenon of RNA interference (RNAi). miRNAs and siRNAs are created in the same way and both mediate the down-regulation of gene expression. But siRNAs work by binding to specific mRNAs and labeling them for destruction by enzymes called Endonucleases. Small nucleolar RNAs (snoRNAs) snoRNAs modify ribosomal RNAs (rRNAs) by orchestrating the cleavage of the long pre-rRNA into its functional subunits (18S, 5.8S and 28S molecules). snoRNAs also add finishing modifications to the rRNA subunits. Small nuclear RNAs (snRNAs) These are constituents of the spliceosome, the cellular machinery that helps to produce mRNA by removing the non- coding regions (introns) of genes and piecing together the coding regions (exons) to be translated into proteins. Some of these snRNAs have been shown to be the functional enzymes in the splicing reaction. 44  UNDERSTANDING TRANSCRIPTION RNA polymerase (RNAp) (Enzyme) DNA RNA  One DNA strand serves as the template  The site of DNA to which RNAp binds is called “Promoter”  For this binding, one needs additional proteins called “Transcription factors”  Promoter contains information that determines which of the two DNA strands is transcribed & the site at which transcription begins.  RNAp moves along template DNA strand from 3’ to 5’, unwounding  A complimentary strand of mRNA grows from 5’ to 3’ 45 Summary: Chain elongation Mechanism during Transcription RNA polymerase moves along the template DNA strand from 3’-5’. As the polymerase progresses, the DNA is temporarily unwound and the polymerase assembles a complementary strand of RNA that grows from 5’ to 3’ direction. RNA polymerase catalyzes the reaction (n = no of nucleotide) RNAn + NTP RNAn+1 + PPi In which (ribo)Nucleoside Tri-Phosphate substrates (NTPs) are cleaved into nucleoside monophosphate as they are polymerized into a covalent chain Pyrophosphatase enzyme PPi 2Pi + Free energy The released energy makes the incorporation of nucleotides irreversible. As the polymerase moves along the DNA template, it in- corporates complementary nucleotides into the growing RNA chain. Once the polymerase has moved past a particular stretch of DNA, the DNA double helix reforms 46  Genetic Code and its properties  One of the first models of the genetic code was presented by the physicist George Gamow, who proposed that each amino acid in a polypeptide was encoded by three sequential nucleotides. In other words, the code words, or codons, for amino acids were nucleotide triplets.  There are 4 possible one-letter words, 16 (42) possible two-letter words, and 64 (43) possible three-letter words. Because there are 20 different amino acids (words) that have to be specified, codons must contain at least 3 successive nucleotides (letters). The triplet nature of the code was soon verified in a number of insightful genetic experiments conducted by Francis Crick, Sydney Brenner, and colleagues at Cambridge University.6  The genetic code is the set of rules by which information encoded in genetic material (DNA or mRNA sequences) is translated into proteins (amino acid sequences) by living cells. 47 47 Genetic Code 48 (Nirenberg & Khorana, 1968) 48  Features of the Genetic Code  It transfers information from mRNA to proteins with high fidelity  It is redundant/degenerate: 61 mRNA triplets code for 20 amino acids  Contains START (AUG) and STOP (UAA, UAG and UGA) codons  Most codons for a given amino acid differ only in the last (third) base of the triplet (Exceptions: Leu, Arg, Ser)  The Genetic Code is nearly universal: correspondence between a nucleotide triplet and an amino acid is identical from viruses to mammals. The rare exceptions are mitochondria and unicellular protozoa.  The universality of the Genetic Code is a result of strong evolutionary pressure: a change in a single codon would alter nearly every protein made by an organism. 49 Example Met (Start) Thr Glu Leu Arg Ser STOP m Peptide 50