Topic 4 Gene expression_CConstantinou_2024_Final.pdf

Topic 4: Gene expression Connection between Genes and Proteins, The Genetic Code, Transcription and Translation Prof Constantina Constantinou [email protected] Monday 19th - Tuesday 20th of February 2024 Learning Objectives (LOBs) 1. Describe the structure and functions of RNA. 2. Describe...

Topic 4: Gene expression Connection between Genes and Proteins, The Genetic Code, Transcription and Translation Prof Constantina Constantinou [email protected] Monday 19th - Tuesday 20th of February 2024 Learning Objectives (LOBs) 1. Describe the structure and functions of RNA. 2. Describe the steps and molecules involved in transcription and RNA processing. 3. Describe the steps and molecules involved in translation. Reading: Chapter 17 Lecture 1 The Genetic Code: Connection between Genes and Proteins From Gene to Protein Central dogma of flow of genetic information DNA Transcription RNA Translation Protein The ‘Central Dogma’ was first proposed in 1956 by Francis Crick, discoverer of the structure of DNA. It is the process by which the instructions in DNA are converted into a functional product. The Flow of Genetic Information The DNA information is stored in the nucleotide sequence The DNA sequence of an organism (genotype) leads to specific traits (phenotype) by directing the synthesis of proteins Phenotype is the result of protein production (gene expression) https://www.vecteezy.com/vector-art/7165604-genotype-versus-phenotype Proteins: the links between genotype and phenotype The Flow of Genetic Information Gene expression: DNA-directed protein synthesis Includes 2 stages: (a) Transcription (a) Translation DNA (a) Transcription RNA (b) Translation Protein Genes specify proteins via transcription and translation Genotype: the genetic make up of an organism (defined by the genes on the DNA) DNA Phenotype: the physical appearance (characteristics) of an organism RNA Genotype Transcription Translation Protein Phenotype Basic Principles of Transcription and Translation RNA: the intermediate between genes and the proteins for which they code Transcription: ▪ The DNA-directed synthesis of RNA ▪ Transcription produces messenger RNA (mRNA) Translation: ▪ The synthesis of a polypeptide which occurs under the direction of mRNA ▪ Translation occurs on ribosomes The Central Dogma of Flow of the Genetic Information Cells are governed by a cellular chain of commands: DNA Transcription RNA Translation Protein The Central Dogma of Flow of the Genetic Information https://microbenotes.com/central-dogma-replication-transcription-translation/ Differences in gene expression between prokaryotes and eukaryotes Prokaryotes: ▪ No nuclear envelope to separate transcription and translation ▪ mRNA produced by transcription is immediately translated without further processing (no RNA processing) ▪ Translation can start before the end of transcription Eukaryotic cells: ▪ The nuclear envelope separates transcription from translation ▪ Transcription: takes place in the nucleus ▪ RNA processing: eukaryotic RNA transcripts are modified to yield the mRNA (final product of transcription) – occurs in the nucleus ▪ Translation: takes place in the cytoplasm The Central Dogma in prokaryotic vs eukaryotic cells https://www.mun.ca/biology/scarr/iGen3_05-09.html Gene expression in prokaryotes TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell Fig. 17-3a-2 Gene expression in eukaryotes Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING Primary transcript (Pre-mRNA) is the initial RNA transcript mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell Fig. 17-3b-3 The Genetic Code How are the instructions for assembling amino acids into proteins encoded into DNA? 4 nucleotide bases There are 20 amino acids, but there are only 4 nucleotide bases (A, T, G, C) in DNA How many bases correspond to an amino acid? 20 amino acids Fig. 17-3b-3 Codons: Triplets of Bases Information flow from gene to protein is based on a triplet code consisting of three consecutive nucleotides Each gene is transcribed in a complementary mRNA containing the nucleotide triplets (the mRNA base triplets = codons) Each codon specifies the addition of a specific amino acid The gene is translated into an amino acid chain forming a polypeptide chain Codons: Triplets of Bases During transcription, 1 of the 2 DNA strands (i.e. the 3’-5’ DNA strand) is used as a template for the synthesis of the complementary RNA strand Template DNA strand in transcription: the 3’-5’ DNA strand ▪ RNA polymerase synthesizes RNA in a 5’-3’ direction using the template DNA strand RNA transcripts are single stranded molecules During translation, codons on the mRNA molecule are read in the 5’-3’ direction by the ribosomes The DNA Template Strand and the DNA Coding Strand Gene 2 DNA molecule Gene 1 Gene 3 DNA Template strand DNA Coding strand (Non-template strand) 4 nucleotide bases: Base pairing: A= Adenine (Purine) C= Cytosine (Pyrimidine) G= Guanine (Purine) T= Thymine (Pyrimidine) A with T C with G Fig. 17-4 DNA Template strand DNA Coding strand These sequences are identical except T in DNA is replaced with U (Uracil) in mRNA and nucleotides contain ribose instead of deoxyribose Direction of Transcription TRANSCRIPTION mRNA Codon TRANSLATION C N Amino acid An mRNA molecule is complementary to its DNA template Fig. 17-4 Example of Transcription DNA template strand 3’ 5’ DNA coding strand 5’ 3’ Direction of Transcription mRNA 5’ 3’ The Genetic Code 64 codons (triplets): ▪ 61 code for amino acids ▪ 3 triplets (UAA, UAG, UGA) are “stop” signals to end translation AUG = Methionine or Start Codon 64 codons but only 20 amino acids Genetic code characteristics: ▪ It is redundant: more than 1 codons can encode for a specific amino acid ▪ It is not ambiguous: each codon can specify only 1 amino acid Codons must be read in the correct reading frame (correct nucleotide groupings) in order for the specified polypeptide to be produced Second mRNA base Table of the genetic code Third mRNA base (3’ end of codon) AUG = start codon or methionine First mRNA base (5’ end of codon) Three stop codons Fig. 17-5 Codons must be read in correct Reading Frame DNA template strand 3’ 5’ DNA coding strand 5’ 3’ mRNA 5’ 3’ Met Lys Phe Gly STOP Codons must be read in correct Reading Frame DNA template strand 3’ 5’ DNA coding strand 5’ 3’ mRNA 5’ 3’ Met Lys Stop Phe Gly STOP Evolution of the Genetic Code The genetic code is nearly universal: shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated after being transplanted from one species to another ▪ Example: genetically modified bacteria ▪ Bacteria can be programmed by the insertion of human genes to produce certain human proteins for medical use (e.g. insulin) (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene Fig. 17-6 RNA Structure and Function RNA= Ribonucleic acid Produced by transcription of the DNA Single stranded molecule - consists of ribonucleotides Ribonucleotides (RNA nucleotides) consist of: ▪ A nitrogenous base (A,G,C or U) ▪ A pentose sugar (ribose) ▪ A phosphate group Nucleotide structure 5’ end Nitrogenous bases Pyrimidines 5'C 3C Nucleoside Nitrogenous base Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA) Purines Phosphate group 5' C Sugar (pentose) Adenine (A) Guanine (G) (b) Nucleotide 3' C Sugars 3’ end (a) Polynucleotide, or nucleic acid Nucleotide = Nucleoside (nitrogenous base+ pentose sugar) + phosphate group Deoxyribose (in DNA) Ribose (in RNA) (c) Nucleoside components: sugars Fig. 5-27 Ribonucleotide Structure In RNA: ▪ Uracil (U) replaces thymine (T) ▪ The pentose sugar is ribose Pentose sugars Nitrogenous bases Deoxyribose (in DNA) Pyrimidines Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA) Purines Adenine (A) Guanine (G) Deoxyribose is lacking one oxygen atom Ribose (in RNA) Differences between DNA and RNA DNA is double stranded, RNA is single stranded The sugar in DNA is deoxyribose, the sugar in RNA is ribose The Nitrogenous bases present in DNA are: – Thymine (T) – Adenine (A) – Guanine (G) – Cytosine (C) The bases present in RNA are: – Uracil (U) – Adenine (A) – Guanine (G) – Cytosine (C) https://www.differencebetween.com/difference-between-dna-and-vs-rna-structure/ Major types of RNA mRNA = messenger RNA ▪ Function: translated into proteins rRNA = ribosomal RNA ▪ Function: part of ribosome structure tRNA = transfer RNA ▪ Function: transfers amino acids to the growing polypeptide chain during translation Transcription Fig. 17-3a-2 Transcription: DNA-directed synthesis of RNA Transcription: DNA-directed synthesis of RNA: ▪ The first stage of gene expression ▪ Catalyzed by RNA polymerase ▪ RNA synthesis similar to DNA replication, except that uracil substitutes for thymine ▪ Starts with binding of RNA polymerase to a DNA region called promoter Molecular Components of Transcription Promoter: ▪ The DNA sequence where RNA polymerase attaches to ▪ Located at the 5’ end (upstream) of the gene Terminator: the sequence signaling the end of transcription in bacteria (termination is different in eukaryotes) Transcription unit: the stretch of DNA that is transcribed Bacteria have only 1 RNA polymerase Eukaryotes have 3 types of RNA polymerases The eukaryotic RNA polymerase involved in transcription is RNA polymerase II DNA Coding strand (Non-template strand) DNA Template strand 3’ DNA Template strand 5’ Transcription unit Transcription RNA polymerase: ▪ Synthesizes RNA using a DNA template strand ▪ Only transcribes 1 of the 2 DNA strands (the 3’-5’ template strand) ▪ Adds nucleoside triphosphates (ΝΤPs) only in 5’ to 3’ direction ▪ Does not require the presence of an RNA primer for adding NTPs Transcription Eukaryotic RNA polymerases: ▪ RNA polymerase Ι: rRNA synthesis (5.8S (60S ribosomal subunit), 18S (40S ribosomal subunit) and 28S (60S ribosomal subunit)) ▪ RNA polymerase ΙΙ: mRNA and snRNAs synthesis ▪ RNA polymerase ΙΙΙ: tRNA and small RNAs (e.g. 5S rRNA (60S subunit)) synthesis Transcription stages: ▪ Initiation: RNA polymerase ΙΙ binds to the promoter ▪ Elongation: Adds NTPs in 5’ to 3’ direction ▪ Termination: The RNA transcript is released from DNA when it reaches the termination of transcription Promoter Upstream Downstream Transcription unit Promoter 5 3 3 5 3 Start point 5 RNA polymerase Template strand of DNA RNA transcript The promoter: includes transcription start point (nucleotide RNA synthesis begins) and extends several dozen nucleotide pairs upstream from the start point. 1. Binding site of RNA polymerase 2. Determination of which DNA strand will be used as template Transcription Transcription unit Promoter 5 3 3 5 3 5 RNA polymerase RNA transcript Direction of Transcription Transcription is unidirectional. DNA template is read 3’-5’. mRNA is produced 5’-3’ always! Template strand of DNA Transcription There are three stages in Transcription: ▪ Initiation ▪ Elongation ▪ Termination Promoter General depiction of transcription for prokaryotes and eukaryotes Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 3 5 Unwound DNA RNA transcript 2. The RNA polymerase moves downstream, unwinding the DNA and elongating in a 5’-3’ direction Template strand of DNA 2 Elongation Bacteria: 1 RNA polymerase Rewound DNA 5 3 3 5 3 5’ Eukaryotes: RNA polymerase II RNA transcript 1. After RNA polymerase binds to the promoter, the DNA strands unwind and the RNA polymerase initiates RNA synthesis 3 Termination 5 3 3. Eventually the RNA transcript is released and the RNA polymerase detaches from the DNA 3 5 5 Completed RNA transcript 3 Fig. 17-7a-4 Nontemplate strand of DNA Elongation RNA polymerase RNA nucleotides 3 3’end 5 5 Direction of transcription (“downstream”) Newly made RNA Template strand of DNA Fig. 17-7b 1. Initiation of Transcription at a Eukaryotic promoter Promoters: signal the initiation of RNA synthesis by binding of RNA polymerase II General transcription factors: proteins that mediate the binding of RNA polymerase II to the promoter and the initiation of transcription Transcription initiation complex: transcription factors + RNA polymerase II, bound to a promoter TATA box: a specific sequence within a promoter responsible for the assembly of the initiation complex in eukaryotes Transcription initiation stages: 1. General transcription factors bind to the TATA box within the promoter 2. RNA polymerase II is recruited and binds to the promoter 1A Promoter eukaryotic promoter includes a TATA box Template 5 3 3 5 TATA box Transcription factors Start point Template DNA strand 2 Several transcription factors must bind to the DNA before RNA polymerase II can do so 3 5 5 3 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex Fig. 17-8 2. Elongation of the RNA Strand Untwisting of the double helix (10 to 20 DNA bases at a time) Addition of nucleotides to the 3’ end of the RNA strand (in 5’-3’ direction) 40 nucleotides per second (in eukaryotes) A gene can be transcribed simultaneously by several RNA polymerases so large amounts of a protein can be produced in a specific period of time 3. Termination of Transcription In bacteria: ▪ The RNA polymerase stops transcription at the end of the terminator (RNA sequence) (transcription termination point) ▪ mRNA is translated without being processed ▪ Translation can start before the termination of transcription In eukaryotes: ▪ RNA polymerase II transcribes a polyadenylation signal (ΑΑUAAA) ▪ The RNA transcript is released 10–35 nucleotides after the polyadenylation signal Βacteria: translation can start before the end of transcription RNA polymerase DNA mRNA Polyribosome 0.25 m Direction of transcription Polyribosome Polypeptide (amino end) Ribosome Polypeptide mRNA (5 end) DNA Fig. 17-3a-2 Lecture 2 Eukaryotic cells modify RNA after transcription Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide Eukaryotic cell Fig. 17-3b-3 RNA processing in eukaryotic cells Eukaryotic cells modify the RNA transcript (primary RNA transcript or premRNA) before translation Τhe mature mRNA is transferred to the cytoplasm after RNA processing RNA processing includes: ▪ Addition of 5΄Cap: a modified nucleotide (m7G) at the 5’ end ▪ Addition of poly-Α tail at the 3΄end (50-200 adenine residues) ▪ RNA splicing: removal of certain segments from the primary transcript Role of RNA end modifications (5’ Cap and poly-A tail): ▪ Facilitate the export of mRNA from the nucleus ▪ Protect mRNA from degradation by hydrolytic enzymes ▪ Help ribosomes attach to the 5‘ end during translation RNA processing Pre-mRNA RNA splicing: removal of certain segments from the primary transcript 5 Cap Addition of 5΄Cap: a modified nucleotide (m7G) at the 5’ end Poly-A tail mRNA Addition of poly-Α tail at the 3΄end (50-200 adenine residues) RNA processing: addition of 5’-cap and 3’ poly-A tail 5 G Protein-coding segment Polyadenylation signal 3 P P P 5 Cap AAUAAA 5 UTR Start codon 5’ CAP is a modified guanine (G) nucleotide Stop codon 3 UTR UTR= untranslated region AAA…AAA Poly-A tail 50-200 Adenine (A) nucleotides Fig. 17-9 RNA Splicing Only a small part of the genome encodes for proteins The larger part of the genome: ▪ Does not code for proteins (non-coding regions) ▪ Has regulatory role, regulates gene expression (e.g. promoters) Introns: the non-coding regions of genes ▪ Have regulatory sequences (regulate gene expression) ▪ Role in RNA splicing Exons: the coding regions of genes ▪ Expressed by translation into amino acid sequences RNA splicing ▪ Removes introns and joins exons ▪ Creates the final mRNA molecule that has a continuous coding sequence Average length of transcription unit along a DNA molecule: 27000 base pairs Primary RNA transcript: 27000 base pairs mRNA: 1200 base pairs Protein: 400 amino acids RNA Splicing of β-Globin, one of the polypeptides of hemoglobin (146 amino acids) 5 Exon Intron Exon Exon Intron 3 Pre-mRNA 5 Cap Poly-A tail 1 30 31 Coding segment mRNA 5 Cap 1 5 UTR 104 105 146 Introns cut out and exons spliced together Poly-A tail 146 3 UTR Introns are cut out and Exons are joined together 5’UTR and 3’UTR are not translated (UTR= untranslated regions) → facilitate translation Fig. 17-10 RNA Splicing RNA splicing is carried out by spliceosomes in the nucleus of eukaryotic cells Spliceosomes: ▪ A large and complex molecular assembly consisting of snRNPs (small nuclear ribonucleoproteins) and other proteins ▪ snRNPs consist of proteins and small nuclear RNAs (snRNAs) (