Gene Structure and Expression - PDF

GENE STRUCTURE AND EXPRESSION WHAT DO WE NEED GENES FOR? We are going to use genes to build proteins through translation and transcription Genes hold the instructions on how to build protein HOW DO WE KNOW THAT GENES ENCODE PROTEINS? 1) (1896): GARROD studied alkaptonuria disease...

GENE STRUCTURE AND EXPRESSION WHAT DO WE NEED GENES FOR? We are going to use genes to build proteins through translation and transcription Genes hold the instructions on how to build protein HOW DO WE KNOW THAT GENES ENCODE PROTEINS? 1) (1896): GARROD studied alkaptonuria disease ○ Metabolic disorder ○ Produce chemical that turns black in air ○ Inherited Alteration in a GENE that encodes the ENZYME that metabolizes this chemical ○ He is the first to make a connection between metabolic disorders and inheritance 2) (1940s): BEADLE AND TATUM studied orange bread mold (NEUROSPORA CRASSA) ○ Mold grows on minimal media ○ Used X-rays to produce nutritional mutants (auxotrophs) ○ HYPOTHESIS: each mutant had defective GENE for ENZYME needed to synthesize a particular nutrient BEADLE AND TATUM EXPERIMENT ARGININE synthesis pathway Each step is controlled by a GENE that encodes an ENZYME for that step  Each of these steps is an enzyme catalyzed reaction If you take wild type bread mold and you grow it on minimal media, it will grow because it can do all of the steps The wild type was compared to the auxotrophs (the ones mutated from X-rays) None of the mutants can grow on minimal media Because the X-ray affects the DNA, therefore, whatever the defect that was in the enzyme, was caused by the defect in the gene. In the first step, the wild type was able to take that precursor and make ornithine and continue the pathway The mutants have a defect in one of those genes The ArgE mutant cannot produce enzyme 1, but if you give it ornithine it can continue the pathway The argF mutant cannot produce enzyme 2, so it can produce ornithine but not citrulline. RELATIONSHIP BETWEEN GENES AND PROTEINS ONE GENE - ONE ENZYME HYPOTHESIS ○ Direct relationship between genes and enzymes ONE GENE - ONE POLYPEPTIDE HYPOTHESIS ○ NOT all proteins are enzymes ○ Functional proteins sometimes contain one or more POLYPEPTIDES ○ Different genes encode each POLYPEPTIDE A prime example is hemoglobin HOW TO GET FROM GENES TO PROTEINS 1) TRANSCRIPTION ○ Nucleotide sequence in DNA is copied into a complementary sequence in an RNA molecule ○ TEMPLATE STRAND of DNA is used to create MESSENGER RNA (mRNA) 2) TRANSLATION ○ Sequence of nucleotides in mRNA molecule specifies amino acid sequence in polypeptide ○ RIBOSOME assembles the amino acid sequence Ribosome that is not membrane bound TRANSCRIPTION AND TRANSLATION Both prokaryotic and eukaryotic organisms do this They both do transcription, using DNA to build an mRNA copy And using the mRNA copy to build proteins In prokaryotes, since there are no membrane bound organelles and nucleus, both translation and transcription happens in the cytoplasm  In eukaryotes, transcription happens inside the nucleus, translation happens outside the nucleus (in the cytoplasm) GENETIC CODE INFORMATION ○ 3 nucleotide bases in DNA or RNA ○ 20 different amino acids in polypeptides CODE ○ One nucleotide → Only 4 combinations ○ Two nucleotides → Only 16 combinations ○ Three nucleotides → 64 combinations 64 is plenty of different combinations to code for the 20 amino acids DNA ○ Three-letter code: TRIPLET RNA ○ Three-letter code: CODON ○ Codon is built during transcription The triplet and codon are complementary to one another. ONE codon encodes ONE amino acid ○ This is done during translation Read 3’ → 5’ THe RNA copy will be read 5’ → 3’ ○ U is substituted for T When you build your polypeptide, you will read N-terminus (the amino group) → C terminus (to the last amino acid with the carboxyl group at the end) FEATURES OF THE GENETIC CODE SENSE CODONS ○ 61 codons specify amino acids ○ Most amino acids specified by several codons (REDUNDANCY) ○ Ex: CCU, CCC, CCA, CCG all specify proline ○ Nucleic acid codes are sequential ○ NO spaces between codons ○ Start codon AUG establishes the READING FRAME START CODON (INITIATOR CODON) ○ AUG ○ First codon recognized during translation ○ Specifies amino acids METHIONINE STOP CODONS (TERMINATION CODONS) ○ These don’t code for any amino acids ○ End of a polypeptide-encoding mRNA sequence Once you reach a stop codon, you stop translating ○ UAA, UAG, UGA CODONS GENETIC CODE IS UNIVERSAL Same codons specify the same amino acids in ALL living organisms and viruses Genetic code was established very early in the evolution of life and has remained UNCHANGED TRANSCRIPTION (DNA → RNA) Info in DNA is transferred to a complementary RNA copy  Similar to DNA replication, except ○ Only ONE DNA strand used as a template ○ Only transcribes the genes ○ RNA POLYMERASE used ○ RNA are single strands ○ Uracil replaces THYMINE In DNA replication, you use both DNA strands In DNA transcription, you separate and only use one strand In DNA replication, you transcribe the whole chromosome → In transcription, you only transcribe genes RNA POLYMERASES NO primers needed to start complementary copy RNA is made in the 5’ → 3’ direction ○ DNA template strand is read 3’ → 5’ Euk - RNA polymerase does NOT bind directly to DNA ○ Needs TRANSCRIPTION FACTORS These are small proteins that bind to the DNA first and then when they bind, the RNA polymerase binds to them Prok - RNA polymerase binds directly to DNA TRANSCRIPTION OVERVIEW Begins as RNA polymerase BINDS to DNA DNA double helix begins to UNWIND RNA polymerase ADDS RNA nucleotides sequentially according to the DNA template Enzyme and completed RNA transcript RELEASE from DNA template ORGANIZATION OF A GENE In a gene, you are going to have 3 major parts: 1) PROMOTER ○ Control sequence initiates transcription ○ Upstream of transcriptional unit ○ Where RNA polymerase binds 2) TRANSCRIPTION UNIT ○ Portion of gene that is copied into RNA 3) TERMINATOR ○ Signals the end of the transcription of a gene ○ RNA polymerase comes off the DNA and the RNA transcript also comes off. ○ DNA comes back together again  The binding of the transcription factor to the TATA box is the first step of transcription TRANSCRIPTION: INITIATION, ELONGATION AND TERMINATION RNA polymerase recognizes the transcription factors and comes on When it comes on, it creates an initiation complex RNA polymerase will separate the two strands of DNA and that means that now that DNA is ready to be transcribed  RNA polymerase is going down the unit and building the new RNA strand In the mouth of the enzyme, the active site, the DNA template strand is with the new RNA (still attached to the template strand) this is called the hybrid RNA-DNA double helix, as RNA polymerase moves down the DNA, the two will eventually separate. INITIATION RNA polymerase II Euk - have TATA BOX in promoter TRANSCRIPTION FACTORS bind to promoter RNA POLYMERASE II binds transcription factors ○ Unwind DNA and begin translation TERMINATION Termination of transcription differs in eukaryotes → POLYADENYLATION signal Prokaryotes have TERMINATORS TRANSCRIPTION OF NON-CODING REGIONS Non-coding genes do not code for protein but instead code for rRNA and tRNA ○ Coding genes are genes that code for proteins Euk - use RNA polymerase III for tRNA and 1 rRNA ○ Use RNA Polymerase I for 3 rRNAs ○ DIFFERENT promoters On the coding genes, we use RNA polymerase II to make messenger RNA  But if you want to make the other RNAS, you are going to use RNA polymerase III Prok - use RNA polymerase II for all transcription ○ SAME promoters MESSENGER RNA PROKARYOTES ○ Coding region flanked by 5’ and 3’ UNTRANSLATED regions (UTRs) EUKARYOTES ○ Coding region flanked by 5’ and 3’ UNTRANSLATED regions (UTRs) ○ Additional NONCODING elements (INTRONS) We need to get rid of introns before translation PRE-MRNA PRECURSOR-MRNA (PRE-MRNA) ○ Must be processed in nucleus to produce translatable mRNA 5’ CAP ○ A guanine-containing nucleotide is added to the 5’ end in the reverse order In the reverse order, it will signal to the ribosome that this is the end → The ribosome has to read the messenger RNA from 5’ to 3’. ○ Reversed GUANINE -containing nucleotide ○ Site where RIBOSOMES attaches to mRNA POLY(A) TAIL ○ 50 to 250 ADENINE nucleotides added to 3’ end by POLY A POLYMERASE ○ Protects mRNA from RNA-digesting enzymes Without the tail, it is prone to degradation INTRONS (only in eukaryotes) ○ non-protein-coding sequences in the pre-mRNA ○ Must be REMOVED before translation EXONS ○ Amino acid coding sequences in pre-mRNA ○ Joined together SEQUENTIALLY in final mRNA HOW DO WE REMOVE INTRONS? Poly(A) polymerase Start with a DNA molecule that still has introns and exons MRNA SPLICING Introns in pre-mRNAs REMOVED SPLICEOSOME ○ pre-mRNA ○ Small ribonucleoprotein particles (snRNP) Small nuclear RNA (snRNA) + several proteins snRNPs - ribonucleoprotein particles ○ Bind to INTRONS ○ LOOP introns out of the pre-mRNA ○ Clip the intron at each exon boundary ○ Join adjacent EXONS together Will ligate them that will join the exons together to form a sequential sequence  RNA part does the cutting → ribozymes Ribozymes → an enzyme catalyzing like structure made of RNA ○ RNA has catalytic activity itself. You can’t let an exon exist in the final product. WHY ARE INTRONS PRESENT? ALTERNATIVE SPLICING ○ DIFFERENT versions of mRNA can be produced Happens most transcriptionally, actual mRNA EXON SHUFFLING ○ Generates new PROTEINS Happens from the genomic level → PROTEIN VARIABILITY = DIVERSITY ALTERNATIVE SPLICING Exons joined in different combinations to produce different mRNAs from the same gene Different mRNA versions translated into different proteins with different functions More information can be stored in the DNA ○ → EFFICIENCY ALTERNATIVE mRNA SPLICING α-tropomyosin in smooth and striated muscle ○ The difference is that alternative splicing occurred between exons ○ Even though they are both isoforms (in the same family), they are different functionally Similar protein but DIFFERENT structure ○ Therefore, DIFFERENT function ○ Notice how exons are still in numerical order EXON SHUFFLING Intron-exon junctions often occur between major functional regions in encoded protein EXON SHUFFLING mixes protein regions or domains into novel combinations ○ Happens in the genomic level ○ You can get the complete reordering of exons ○ Allows quicker and more efficient evolution of new PROTEIN TRANSLATION (MRNA → PROTEIN) Assembly of amino acids into polypeptides Occurs on RIBOSOMES P, A and E sites on ribosome used for stepwise addition of amino acids to polypeptide as directed by mRNA TRANSLATION OVERVIEW A site is first → then P site → then E (exit) site The whole thing is called a translational assembly tRNAs TRANSFER RNAs (tRNA) ○ Bring specific amino acids to ribosome ○ Cloverleaf shape ○ Bottom end of tRNA contains ANTICODONS sequence that pairs with codon in mRNAs ○ Top end contains AMINO ACIDS tRNA STRUCTURE Clover leaf structure Read 3’ → 5’ ○ If the codon is read 5’ → 3’, the anticodon is read 3’ → 5’ for complementation DO WE HAVE 61 tRNAs? WOBBLE HYPOTHESIS ○ 61 different sense codons do NOT require 61 different tRNAs First two nucleotides of anticodon and codon must match EXACTLY → the first two are important (essential) Third nucleotide has more FLEXIBILITY ○ This is some sort of protection against mutations. Ex: tRNA carrying glutamine ○ Matches codons CAA and CAG AMINOACYLATION Adds amino acid to tRNA ○ AMINOACYL-tRNA (Amino acid linked to tRNA) ○ AMINOACYL-tRNA SYNTHETASES catalyze reaction NEED ATP TO DO THIS.  Three major binding sites ○ ATP binding site ○ Amino acid binding site ○ Anticodon binding site First, you need to recruit ATP and the amino acid You are going to remove a phosphate to get an AMP that is linked to the amino acid Then you can recruit the right tRNA molecule that can fit into the anticodon site. ○ The reaction lasts long enough if the right one links, while if the wrong one links it will quickly disassociate from the enzyme. Most of the energy released from ATP hydrolysis is kept in the peptide bond to link amino acids in translation. RIBOSOMES Made of ribosomal RNA (rRNA) and proteins ○ Two subunits: Large and small  In terms of the critical site, the growing peptide strand is going to come from the P site Amino acid comes in from the A site. TRANSLATION STAGES 1) INITIATION ○ Ribosomes assembled with mRNA molecule and initiator METHIONINE-tRNA ○ For initiation to happen, it relies on a certain codon to start 2) ELONGATION ○ Amino acids linked to tRNAs added one at a time to growing polypeptide chain 3) TERMINATION ○ New polypeptide released from ribosome ○ Ribosomal subunits separate from mRNA INITIATION 1) Initiator tRNA (Met-tRNA) binds to small subunit 2) Complex binds to 5’ cap of mRNA, scans along mRNA to find AUG start codon 3) Large ribosomal subunit binds to complete initiation ○ Met-tRNA is in the “P” site Unique because no other amino acids with its tRNA will first go to the P site → they will all go to A site first In prokaryotes, there is NO 5’ cap so bind directly to the site just before AUG. Only one to go directly to P SITE  Establishing the reading frame ELONGATION Aminoacyl-tRNA matching the next codon enters A SITE  PEPTIDYL TRANSFERASE catalyzes formation of first peptide bond and cleaves tRNA in P SITE Ribosome moves along mRNA to next codon ○ Empty tRNA moves from P-site to E-site, then released ○ Newly formed peptidyl-tRNA moves from A-site to P-site ○ A site empty again TERMINATION Begins when A SITE reaches stop codon Release factor (RF) or TERMINATION FACTOR binds to A site Polypeptide chain released from P SITE Remaining parts of complex separated  Happens one at a time. POLYSOMES Multiple ribosomes can SIMULTANEOUSLY translate a single mRNA This increases efficiency as there is multiple translation happening at once (only in eukaryotes) SIMULTANEOUS TRANSCRIPTION AND TRANSLATION Can occur in prokaryotes (no nuclear envelope)  Prokaryotes couple translation and transcription ○ They can do transcription at the same time they do translation POLYPEPTIDE PROCESSING Processing reactions convert polypeptides into finished form ○ REMOVAL of one or more amino acids from the protein chains ○ ADDITION of organic groups Can also have inorganic modifications ○ FOLDING guided by chaperones ○ Alternative pathways to different mature polypeptides WHAT ABOUT PROTEINS IN THE SECRETORY PATHWAY? Proteins are distributed within cells by SORTING SIGNALS Signals are coded in DNA, appear when protein is made Ex: insulin → needs to find its way outside of the cell. ○ The signal sequence is found at the end of the protein sequence, but it is encoded for in the nucleus. SORTING SIGNALS IN ER Proteins sorted at rough endoplasmic reticulum SIGNAL PEPTIDE (SIGNAL SEQUENCE) ○ At beginning of polypeptide chain SIGNAL RECOGNITION PARTICLE (SRP) ○ Binds to signal peptide ○ Stops translation → temporary stalls elongation because it needs to shuttle that whole complex to the ER  SRP RECEPTOR ○ SRP binds to protein receptor in ER membrane ○ Ribosome BOUND onto ER membrane ○ Growing polypeptide pushed INSIDE ER lumen Can re-initiation elongation SIGNAL PEPTIDASE ○ Removes signal sequence Translation continues until polypeptide COMPLETED SIGNAL MECHANISM IN ER Once the polypeptide sequence is long enough that it starts to poke out of the ribosome, SRP can bind to the SRP receptor and cleave the single peptide. Once the polypeptide is released, it is now dissolved in the lumen of the rough endoplasmic reticular and then secreted out of the cell. MUTATIONS Changes in genetic material Base-pair mutations change DNA TRIPLET ○ Results in change in mRNA CODON ○ May lead to changes in the AMINO ACID sequence of the encoded polypeptide 1) “MISSENSE MUTATION” - changes a sense codon to a different sense codon 2) “NONSENSE MUTATION” - changes a sense codon to a stop codon → or could change it to worst 3) “SILENT MUTATION” - changes one sense codon to another sense codon that specifies the same amino acid  4) “FRAMESHIFT MUTATION” - Base-pair insertion or deletion alters the reading frame after the point of the mutation ○ If you mutate the first amino acid (or the first nucleotide) within the codon, you are going to completely change the sense codon in the final mRNA which codes for a different amino acid  ○ A pair is changed to a stop codon, so the polypeptide is not complete.  SICKLE-CELL DISEASE Caused by a SINGLE MISSENSE mutation MUTATIONS - SPONTANEOUS OR INDUCED “SPONTANEOUS MUTATIONS” - errors during DNA replication or repair “INDUCED MUTATIONS” - physical, chemical and biological agents (MUTAGENS) generate mutations → MUTAGENESIS ○ MUTAGENESIS - Creating mutations ○ E.g., X-rays, UV radiation, 5-bromouracil INDUCED MUTATIONS - 5-BROMOURACIL “5-BROMOURACIL” - analogue of thymine but has bromine instead of methyl group ○ Can form double H-bond with adenine ○ Can form triple H-bond with guanine ○ Can lead to substitution mutation ○ ○ ○ There are sites where you won’t get an effect since the DNA can retain the normal base pairing, or it can shift to its other form where it adheres to the 5BU and creates a new nucleotide pairing. PUTTING IT INTO PERSPECTIVE 1) What is the connection between DNA, RNA, and protein? 2) What is the process of transcription? 3) What is the process of mRNA processing? 4) What is the process of translation? 5) What can happen when there are mutations?