Unit III Lecture.pdf

Unit III: Gene Expression Clarissa Therese M. Mordeno, DVM Unit Objectives Explain DNA Replication Explain the Central Dogma of Biology Explain protein synthesis process  Review on Nucleic Acids DNA Replication Unit Topic...

Unit III: Gene Expression Clarissa Therese M. Mordeno, DVM Unit Objectives Explain DNA Replication Explain the Central Dogma of Biology Explain protein synthesis process  Review on Nucleic Acids DNA Replication Unit Topic Transcription Outline Central Dogma of Molecular Biology Translation Regulation of Gene Expression Recall: DNA Meaning: Deoxyribonucleic acid Function: carries the genetic code Shape: double helix Location: Chromosomes within the nucleus (most) Nucleotide DNA sugar: deoxyribose DNA Nitrogenous Bases DNA Base pairings: A- T C-G DNA Replication Occurs during S phase of Interphase It is the biological process of producing two identical replicas of DNA from one original DNA molecule Replication is a semiconservative process 3 Models of DNA Replication: 1. Semiconservative DNA replication - produces two DNA helices in which each helix contains one new strand and one old strand 2. Conservative DNA replication - produces two DNA helices, out of which one contains entirely old DNA while the other contains entirely new DNA 3. Disruptive DNA replication - produces two DNA helices in which each strand has alternating segments of old and new DNA Note: Out of the three models theorized, only the Semiconservative model of DNA Replication was elucidated by Matthew Meselson and Franklin W. Stahl in May 1958 through the Meselson-Stahl https://www.youtube.com/watch?v=7-tnuAqEp9g Experiment (1957-1958) DNA Replication Important Steps 1. Pre-initiation – recognition of replication origins 2. Initiation – Licensing of origins of replication to create the replication fork 3. Elongation – synthesis of the new DNA strands as the replication forks progress away from the origins of replication 4. Termination – converging of replication forks 5. Telomere replication/synthesis Origins of DNA Replication Prokaryotic DNA: Replication proceeds bidirectionally from a single origin of replication on the prokaryotic circular chromosome Replication of an E.coli DNA Origins of DNA Replication Prokaryotic DNA: Replication proceeds bidirectionally from a single origin of replication on the prokaryotic circular chromosome Eukaryotic DNA: Replication proceeds bidirectionally from hundreds or thousands of origins of replication on each of the linear eukaryotic chromosomes. Replication of a eukaryotic chromosome. Blue lines are parental strands. Red lines are newly synthesized strands. Eukaryotic Origins of Replication  Replication origin I. Pre-initiation Origin Recognition Complex (ORC) recognizes replication origins and Helicase loading tags these as sites of initiation of proteins replication ORC recruits helicase loading proteins and together, recruit the Helicase helicase enzymes (MCM) These make up the pre-replication complex (Pre-RC) Presence of pre-RC at an origin Pre-replication “licenses” that origin for replication complex II. Initiation Ddt4 dependent kinase (Ddk) and Cyclin dependent kinase (Cdk) activate the pre-RC This results in the assembly of additional replication proteins/enzymes at the pre-RC 1. DNA Polymerase δ - delta 2. DNA Polymerase ε - epsilon 3. DNA Polymerase α - alpha /primase complex II. Initiation Helicase is also activated and functions by unzipping the DNA strands DNA Polymerase α/primase complex synthesizes an RNA DNA Pol α primer and briefly extends it /primase RNA primer contains about 10 ribonucleotides II. Initiation Helicase is also activated and functions by unzipping the DNA strands DNA Polymerase α /primase complex synthesizes an RNA primer and briefly extends it RNA primer contains about 10 ribonucleotides Topoisomerase prevents supercoiling of the DNA ahead of helicase III. Elongation Clamp loader (RF-C) and sliding clamp (PCNA) assemble at the primer- template junctions Polymerase switching occurs: DNA Pol α/primase complex dissociates Replaced by DNA Pol δ or ε These extend the RNA primer  III. Elongation Replication origin DNA polymerases can only synthesize DNA in a 5’ →3’ direction and needs a –OH end to bind Leading strand (DNA Pol δ) - formed by continuous copying of the parental strand that runs 3’ → 5’ toward the replication fork. Lagging strand (DNA Pol ε) - formed by discontinuous copying of the parental strand that runs 3’ → 5’ away from the replication fork. - Okazaki fragments are formed in the lagging strands III. Elongation DNA Polymerase δ FEN1+RNaseH FEN1 and RNaseH remove RNA primers at the start of each leading strand and at the start of each Okazaki fragment DNA Polymerase ε DNA Polymerase α then fills Polymerase α the gaps with the corresponding DNA DNA ligase forms bonds between the sugar-phosphate backbones  IV. Termination occurs when converging ]- replication forks meet on the Termination zone same stretch of DNA topoisomerase Helicase enzymes and Topoisomerase is leaves as helicase they run out of space helicase There is a problem… DNA Polymerase α enzyme requires a free 3’-OH end on which to initiate synthesis Gaps left at the 5’ ends of the newly synthesized DNA cannot be filled by DNA 5’-P 3’-OH 5’-P 5’-P Polymerase because no free 3’-OH groups are available for the initiation of synthesis 5’-P 5’-P V. Telomeric Replication/Synthesis Telomeres maintain chromosomal stability and prevent chromosomal degradation Telomeric DNA in eukaryotes consists of many short, repeated nucleotide sequences Telomerase enzyme maintains of the length of telomeres by addition of GT-rich repetitive sequences V. Telomeric Replication/Synthesis Telomerase enzyme components: 1. TERC (Telomerase RNA component) - Catalytic subunit 2. TERT (telomerase reverse transcriptase) - “guide” to proper attachment of the enzyme  V. Telomeric Replication/Synthesis Template strand New strand Reverse Transcription – DNA is synthesized from RNA Telomere and Aging Not enough telomerase is available all the time for Telomeric Synthesis Telomeres grow shorter as we age Progressive telomerase shortening leads to senescence/apoptosis  A model developed by Francis Crick in The Central Dogma 1958, which demonstrates that genetic of Molecular Biology information flows from DNA to RNA, to make a functional product protein RNA Meaning: Ribonucleic acid Function: messenger Shape: Single-stranded Location: Nucleus and cytoplasm RNA Nucleotide RNA Nitrogenous Bases RNA Base pairings: A- U C-G & nucleus 3 Types of RNA 1. mRNA – messenger RNA 2. rRNA – ribosomal RNA 3. tRNA – transfer RNA  Messenger RNA (mRNA) Carries information from the DNA in the nucleus to the ribosomes in the cytoplasm Serves as the “template” for protein synthesis Transcribed from one strand of DNA and is later translated at the ribosome into a polypeptide Contains codons that codes for a specific amino acid Ribosomal RNA (rRNA) Makes up part of the ribosomes that serve as the site of protein synthesis Together with other proteins, form the ribosome Support the other participants of protein synthesis and help to catalyze formation of bonds between amino acids. Transfer RNA (tRNA) Carries amino acids to the ribosome during translation to help build an amino acid chain Small, single-stranded RNA molecules Has a sequence of 3 bases at one loop (anticodon) Complementary base-pairing of the tRNA anticodon with the mRNA codon brings the correct amino acid (AA) to the ribosome for protein construction Transfer RNA (tRNA) Codon A codon is a trinucleotide sequence in mRNA that specifies for a specific amino acid The Genetic Code Table  Properties of the Genetic code The genetic code is composed of nucleotide triplets The genetic code is degenerate The genetic code is non-overlapping The genetic code is comma-less The genetic code is nearly universal The genetic code is colinear There is polarity in the code The genetic code contains start and stop codons. The Genetic Code Table “The genetic code is degenerate.” - It is redundant - Each of the 20 common amino acids has at least one codon - Many amino acids have numerous codons - Ex. the codons UCU, UCC, UCA, UCG, AGU, and AGC all specify the amino acid Serine The Genetic Code Table “The genetic code is non-overlapping.” each nucleotide is used only once “The genetic code is comma-less.” there are no breaks or markers to distinguish one codon from the next “The genetic code is nearly universal” The same codon specifies the same amino acid in almost all species studied however, some differences have been found in the codons used in mitochondria The Genetic Code Table “The genetic code is colinear.” the order of codons in the mRNA determines the order of amino acids in the encoded protein. “There is polarity in the code.” the code is always read in a fixed direction 5’ 3’ “The genetic code contains start and stop codons.” Start codon – AUG (Methionine) Stop codons – UGA, UAG, UAA Proteins The most important and ubiquitous macromolecules of the cell Most proteins contain all or most of the 20 amino acids (AA) Polymers of AA are known as peptides, polypeptides, and proteins The primary structure of a protein is the result of the order of the nucleotides in the DNA of the gene Protein (Insulin) Genes Units of hereditary information that occupies a fixed position on a chromosome. A sequence of nucleotides in DNA or RNA that encodes the synthesis of a gene product, either RNA or Protein GENE 1 GENE 2 GENE 3 Different Initiation Points Create Overlapping Genes Overview of the Gene Expression Process I. Transcription Transcription Transfer of information from the DNA to the RNA One strand of DNA that makes up a gene called the non-coding strand acts as a template for the synthesis of a complementary RNA strand by an enzyme RNA Polymerase. Once mRNA is formed, it leaves the nucleus through the nuclear pore and attaches to a ribosome Parts of a Eukaryotic Gene Promoter sequence/region Transcriptional initiation region Located near the 5’ end RNA polymerase and transcription factors bind in this region Exons Expressed (coding) sequences Portion of a gene that codes for amino acids Introns Intervening (non-coding) sequences In RNA processing, introns are excised, and exons are spliced together Terminator Sequence Defines the end of a transcriptional unit Initiate the process of releasing the newly synthesized RNA from the transcription machinery Enhancers Sequences that unction in the stimulation of the transcription rate Silencers DNA sequence capable of binding transcription regulation factors Silencers prevent genes from being expressed as proteins Steps in Transcription (mRNA Synthesis) 1.Initiation 2.Elongation 3.Termination 4.mRNA maturation/modification I. Initiation Promoter regions contain conserved sequences: 1. TATA (Hogness) box Sequence: TATATAA located about 25 base pairs upstream (-25) from the transcription start site. 2. CAAT box Sequence: GG(T/C)CAATCT frequently found about 70 base pairs upstream from the start site 3. GC-rich regions occur between -40 and -110. I. Initiation Transcription factors recognize the basal/core promoter region RNA polymerase II (RNAPII) then binds and forms the transcription initiation complex DNA is locally converted from its double stranded form to an open structure, exposing the template strand. II. Elongation RNAPII initiates RNA synthesis A primer is not required Active center The DNA template is copied in the 3′ to 5′ direction, and the RNA chain grows in the 5′ to 3′ direction Abortive transcription occurs until the transcript contains 11 ribonucleotides Template strand vs. Non-template strand Template strand/ antisense strand/ noncoding strand Non-template strand/ sense strand/ coding strand III. Termination RNAPII continues transcription until the polyadenylation signal sequence (AAUAAA) is transcribed Cleavage governed by: CPSF - Cleavage and polyadenylation specificity factor complex CStF - Cleavage stimulatory factor complex III. Termination 5′-exonuclease (called Xrn2 in humans) digests the remaining transcript 5′-exonulease “catches up” to RNA Polymerase II RNA Polymerase II destabilizes and disengages from the DNA template strand Transcription is terminated Pre-mRNA is produced IV. Maturation/Modifications eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated Pre-mRNAs are first coated in RNA-stabilizing proteins; these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus 1. 5’ capping 2. 3’ Poly-A tail 3. Pre-mRNA splicing IV. mRNA Maturation/Modifications 1. 5’ capping - 7-methylguanosine cap is added to the 5' end of the growing transcript – this protects the pre-mRNA from degradation. 2. 3’ Poly-A tail - poly-A polymerase then adds a string of approximately 200 Adenine residues, called the poly-A tail - further protects the pre-mRNA from degradation 3. Pre-mRNA splicing - introns are excised, then exons are spliced together RNA Polymerases Three different RNA polymerases transcribe the nuclear DNA of eukaryotes: RNA polymerase I Found in nucleolus synthesizes precursors of rRNA RNA polymerase II Found in nucleoplasm synthesizes precursors of mRNA RNA polymerase III Found in nucleoplasm synthesizes small RNAs (rRNA, tRNA) II. Translation TRANSLATION The process of converting the information housed in mRNA into the protein sequence Occurs in the cytoplasm mRNA combines with ribosomes, which contain rRNA Many ribosomes can be attached simultaneously to a single molecule of mRNA, forming a polysome. tRNA Charging Also called tRNA aminoacylation Before translation can proceed, the tRNA molecules must be chemically linked to their respective amino acids Enzyme aminoacyl tRNA synthetase directs this process Aminoacyl tRNA synthetase is highly specific (Ester bond) Parts of the Ribosome Aminoacyl (A) site Peptidyl (P) site Exit (E) site Steps in mRNA Translation 1. Initiation 2. Elongation 3. Termination I. Initiation Eukaryotes have a “Cap-dependent translation” Assembly: The following assemble at the 5’ cap or the CCRCCAUG G 7-methylguanosine cap (m7G): 1. Small ribosomal subunit 2. Eukaryotic initiation factors (eIFs) 3. Initiator tRNA (with bound Methionine) Scanning: This assembly slides CCRCCAUG G Kozak sequence along the mRNA searching for a start codon I. Initiation tRNAMet binds to the start codon eIFs are released and the small ribosomal subunit combines with the large subunit to create the initiation complex Functions of eIFs primarily blocks the A site from being bound to a tRNA inhibit the small subunit from associating with the large subunit prematurely Stabilizing tRNAMet at the P site II. Elongation Eukaryotic elongation factors (eEFs) transports the 2nd tRNA into the A site The 2nd tRNA bears the amino acid corresponding to the second codon sequence II. Elongation Ester bond between the P site tRNA and its amino acid is hydrolyzed Amino acid in the P site links to the amino acid in the A site by a peptide bond II. Elongation The uncharged tRNA moves to the E (Exit) site Translocation: The entire mRNA-tRNA-aa2-aa1 complex then shifts in the direction of the P site by a distance of 3 nucleotides shifts This causes a ratchet-like movement of the small subunit relative to the large subunit III. Termination e e Happens when a stop codon in the mRNA (UGA, UAG, and UAA) enters the A site Also called nonsense codons Recognized by eukaryotic release factors (eRFs) e III. Termination e e eRF1 or eRF2 bind to the A site and stimulate hydrolysis of the polypeptide from the tRNA eRF3 binds to the ribosome, and the tRNA is released from the P site e The ribosome dissociates into its subunits e e Parts of the Ribosome Aminoacyl (A) site where charged tRNA with its amino acid enters Peptidyl (P) site Contains tRNA attached to a peptide chain Exit (E) site Regulation of Gene Expression Transcription Regulation Key components: 1. Promoter region 2. Enhancers/Silencers 3. Transcription factors Activators - increases the rate of transcription Repressors – turns off or reduce gene expression Introns 1. Allows some genes to encode for more than one protein product through alternative splicing 2. Evolution of genes and production of new proteins by allowing the phenomenon known as exon shuffling 3. Function as noncoding RNA (e.g. microRNA) 4. Can regulate transcription by containing sequence elements such as enhancers or silencers Alternative Splicing Exon shuffling Histone Modifications - Changing of histone composition to “open” the chromatin for RNA Polymerase to start transcription - Through acetylation, methylation, or phosphorylation References: Klug, W.S., et. al. (2019). Concepts of genetics. 12th ed. Pearson Education, Inc. USA Lieberman, M. & Ricer, R. (2014). Biochemistry, Molecular Biology & Genetics. Lipincott Williams & Wilkins. USA\ Eukaryotic Transcription. (2012). Retrieved from https://pressbooks-dev.oer.hawaii.edu/biology/chapter/eukaryotic- transcription/ Eukaryotic Transcription - Initiation of Transcription in Eukaryotes. Retrieved from https://shorturl.at/65ucI