Gene & Proteins Chapter 15 PDF

Chapter 15 Gene & Proteins  Pt. 1 Genetic Code  Learning Objectives Explain the order of events for the central dogma. Describe Beadle & Tatum’s experiment. Define the term gene. Loading… Apply the genetic code it a sequence of...

Chapter 15 Gene & Proteins  Pt. 1 Genetic Code  Learning Objectives Explain the order of events for the central dogma. Describe Beadle & Tatum’s experiment. Define the term gene. Loading… Apply the genetic code it a sequence of DNA/mRNA. Compare/Contrast Eukaryote & Prokaryote Transcription/Translation. Describe the genetic code, how its used, and its purpose. Define reading frame.  The Central Dogma Cellsare governed by a cellular chain of command that dictate the flow of genetic information. Replication DN DNA RNA PROTEIN Transcription Translation ↑ writing something nucleic acid - protein that is already present nucleic acid - > nucleic acid  Genes (& Proteins) 2 % of total genome. A specific sequence of nucleotides on a strand of DNA. They typically lead to the production of a specific protein product. This can/does lead to the development of a specific trait. Like blood type There are 1,361 genes on chromosome 20! Loading… 5’- ATGCCGATTGAT-3’ 3’- males only TACGGCTAACTA-5’ The smallest gene in the human genome is the SRY gene… 828 nucleotides long (204 amino acids)  Beadle & Tatum One gene= One enzyme hypothesis They developed this hypothesis that stated each gene encodes for a single enzyme. Based on this, they surmised that each gene would influence a specific step in a metabolic pathway. Beadle & Tatum – Neurospora crassa Utilized bread mold (a fungus) to develop their hypothesis! They produced genetic mutants of this fungi using X-rays. Complete medium = all AA, CO2 source, inorganic salts Minimal medium = CO2 source & inorganic salts only Ort Cit  Gene A Gene B Gene C * Spoiler alert, there are way more than 20 amino Enzyme A Enzyme B Enzymeacids C Precursor Ornithine Citrulline Arginine Ort Cit  What we know now… Beyond the one gene – one enzyme hypothesis: Many genes encode for proteins other than enzymes. Many proteins exist and are needed that are not enzymes. (Think transport, signaling, structure, etc.) Some genes encode for only part (subunit) of a protein. (not the whole protein) Remember, many proteins are composed of multiple different polypeptides. (think quaternary) A different gene encodes for each of those different polypeptides.. Some genes encode for non-coding RNAs (rRNA, tRNA, siRNA, miRNA, snRNA, etc.). Many genes have more than 1 exon and can be processed differently to develop different proteins. (alternative splicing)  The Genetic Code… Our genetic information is stored in our sequence of nucleotides. This sequence is decoded by interpreting that sequence in non-overlapping base triplets called codons! Well… There are 20 amino acids that we find in proteins, therefore… We need at least 20 different codes to read. Loading… This means that our genetic code with be redundant! Multiple codons can code for the same amino acid. One amino acid can have several codons that make it! Additionally, we have several stop codons and a start codon.  Evolution of the Genetic Code The Genetic Code is nearly universal! Remember every living organism utilizes DNA, which means they all utilize the same nucleotides A/T/C/G …. U if we also look at their use of RNA! There are very few exceptions to this conservation of the genetic code (i.e. the amino acid code) This means the most organisms utilized the same set of amino acids to build their many different proteins. This provides evidence for a common origin of life on Earth. Suggests life likely evolved from an ancestral organism in which the same genetic code was utilized.  Reading the Genetic Code A gene can be interpreted by reading the codons… however… You must read these codons in the correct reading frame. This refers to which nucleotide starts the first codon of the coding region of the gene. It will always start with the start codon (AUG) which encodes the amino acid Methionine. The Reading Frame of a gene 3’-ATACGGCTAAGCCTGAACGTA-5’ 5’-ATAGCCGATTCGGACTTGCAT-3’ 5’-A UAG CCG AUU CGG ACU UGC AU-3’ 5’-AU AGC CGA UUC GGA CUU GCA U-3’ 5’-AUA GCC GAU UCG GAC UUG CAU-3’ OR 5’-A UGC AAG UCC GAA UCG GCA UA-3’ 5’-AU GCA AGU CCG AAU CGG CAU A-3’ 5’-AUG CAA GUC CGA AUC GGC AUA-3’  Gene Expression The process by which DNA directs protein synthesis. Includes 2 major processes: 1. Transcription The synthesis of RNA under the direction of DNA Produces messenger RNA (mRNA) Produces the template for translation 1. Translation The actual synthesis of a polypeptide Which occurs under the direction of mRNA Occurs on ribosomes  Gene Expression Prokaryotes Eukaryotes NO NUCLEUS!!! Transcription & mRNA modification This means transcription & translation occur in the nucleus. take place in the cytoplasm. Translation occurs in the cytoplasm. They occur in the same place at the same time! Ribosome Unique Prokaryote Feature of Gene Expression Prokaryote Gene Expression happens solely in the cytoplasm. This means transcription and translation occur in the same location. Additionally, Prokaryotes do not require RNA transcript modification. This means their RNA transcripts can be translated immediately after being transcribed. Because of these 2 points, a Prokaryote’s RNA transcript can be translated as transcription is progressing. Multiple polymerases can transcribe a single gene* Numerous ribosomes can concurrently translate the mRNA transcripts into polypeptides.* This can allow a specific transcript and/or a specific protein to rapidly reach high concentrations in a cell.  Pt. 2 Transcription  Learning Objectives Explain the order of events during eukaryotic pre-mRNA processing. State the relationship between exons and protein structure. Statethe sequence of the translation start codon and all three translation stop codons. Using the Genetic Code DNA RNA During Transcription The gene sequence determines the sequence of bases along the length of a mRNA molecule. RNA is comprised of G, C, A, and U Thymine is substituted for uracil in RNA (G:::C and A::U) Occurs in the nucleus of eukaryotic cells (cytoplasm for prokaryotes) RNA polymerase is the enzyme that carries our transcription. Synthesizing RNA: Initiation Promoter Sequence A sequence of DNA that serves as a recognition and recruitment site for Transcription factors and RNA polymerase. Transcription Factors Proteins that aid in the initiation and regulation of transcription RNA polymerase Synthesizes the RNA transcript Breaks the hydrogen bonds between DNA strands and links together RNA nucleotides Same base pairing rules as DNA, except… RNA uses uracil in replace of thymine. RNA polymerase can only add nucleotides onto the 3’ end of the growing RNA transcript. Several types (I, II, and III) Synthesizing RNA: Elongation The production of the RNA transcript. RNA Polymerase Unwinds DNA to access the template strand. Only exposes ~10-20 DNA nucleotides at a time. Connects RNA nucleotides using DNA as a template. Produces the RNA transcript in a 5’ to 3’ direction Typically producing the RNA transcript at ~40 nucleotides per second. Synthesizing RNA: Elongation Direction of transcription Synthesizing RNA: Termination Terminator Sequences A sequence of DNA at the end of a gene that is transcribed and signals the RNA transcript is complete. Termination Variation Prokaryotes RNA polymerase reads through a “termination sequence” This cause the RNA polymerase to dissociate from the DNA. The RNA is immediately ready for translation….boom! Eukaryotes RNA polymerase reads through a special termination sequence known as a Polyadenylation sequence (AAUAAA) The end of RNA transcript is then bound by proteins causing the RNA polymerase to dissociate from the DNA. After termination, the RNA needs additional processing!!!  Summary of Transcription Unwound DNA  Eukaryotic Post-transcriptional Pre-mRNA Processing  1 & 2… Caps and Tails The ends of the mRNA transcript (5’ and 3’ ends) are modified in specific ways. 5’ end This end of the mRNA transcript receives a modified guanine nucleotide. Known as the 5’-methylguanosine cap 3’ end This end of the mRNA transcript receives a poly-adenosine tail. Known as the 3’ poly-A tail This is a string of A nucleotides that can vary in number depending on the transcript, cell type, and organism. A modified guanine nucleotide 50 to 250 adenine nucleotides added to the 5’ end added to the 3’ end TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA Protein-coding segment Polyadenylation signal 5’ 3’ mRNA G P P P AAUAAA AAA…AAA Ribosome TRANSLATION Start codon Stop codon 5’ Cap 5’ UTR 3’ UTR Poly-A tail Polypeptide  3… RNA splicing The process of removing introns and joining together included exon sequences to form a mature mRNA transcript. Ensures that only coding sequences are translated (exons). Removes introns (noncoding sequences) and joins exons. This is accomplished through the use of specialized proteins complexes known as spliceosomes TRANSCRIPTION DNA Spliceosomes – complexes made of protein and catalytic RNA (ribozymes). RNA PROCESSING Pre-mRNA mRNA RNA transcript (pre-mRNA) 5’ Ribosome Exon 1 Intron Exon 2 TRANSLATION Protein snRNA Other proteins Polypeptide snRNPs Intron 5’ Exon Intron Exon Exon 3’ Spliceosome Pre-mRNA 5’ Cap Poly-A tail 5’ 1 30 31 104 105 146 Coding Introns cut out and segment exons spliced together Spliceosome components Cut-out mRNA 5’ Cap Poly-A tail mRNA intron 1 5’ 146 Exon 1 Exon 2 5’ UTR 3’ UTR So… why Introns? Allow for Alternative Splicing. The inclusion of differing sets of exons for differing mature mRNA produced from the same gene. Introns provide alternative cut sites for this. Polypeptides within proteins often have discrete Gene DNA structural and functional regions called domains. Exon 1 Intron Exon 2 Intron Exon 3 In many cases, each exon can encode for a different domain. Transcription Loading… RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide So… why Introns?  Pt. 3 Translation & Protein Synthesis Ribosomes RNA: mRNA, tRNA, rRNA miRNA, siRNA PROTEIN Translation  Learning Objectives Explain the function of tRNA synthetases and why there are 20 of them Draw a diagram of a ribosome that includes all the binding sites and describe what binds at those sites Explain how ribosomes translate the information in an mRNA into a protein Explain the process of translation termination State where all translation is initiated Explain why some proteins are translated on ribosomes bound to the ER, and how these ribosomes are localized to the ER  Ribosomes Using the Genetic Code RNA: mRNA, tRNA, rRNA, miRNA, siRNA PROTEIN During Translation The mRNA sequence determines the sequence of amino acids in the primary structure of the polypeptide. This is considered RNA-directed synthesis of a polypeptide.  TRANSCRIPTION DNA mRNA The Components Ribosome TRANSLATION Polypeptide of Translation Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C A C G Anticodon A A A U G G U U U G G C 5’ Codons 3’ mRNA  The Molecular Components -tRNA tRNA – transfer RNA tRNA molecules are not all identical, however they all:. Carry a specific amino acid on 1 end.. Have an anticodon on the other end.. Single RNA strand that is about 80 nucleotides long.. Utilize a specific Aminoacyl-tRNA synthetase to attach its amino acid.  The Molecular Components - Ribosome Protein and rRNA complex that facilitates the reading of mRNA and production of the corresponding polypeptide Achieved through the paring of mRNA codons with tRNA anticodons. Consists of 2 ribosomal subunits (these vary between prokaryotes and eukaryotes) Has 3 binding sites for tRNA  The Molecular Components -mRNA The molecule that directs the recruitment of tRNA molecules and production of the polypeptide. Very specific sequence of RNA, unique to the polypeptide it will be used to create. mRNA is bonded to the small subunit of the ribosome Read in a 3 base codons in a 5’ to 3’ fashion. AUG is the start codon in every mRNA. Each codon in the mRNA is bonded to the anticodon of the tRNA The Molecular Components -Polypeptide The product of Translation! Produced through the assembly of amino acids bonded together in a specific sequence. Achieved by the interaction of one tRNA in the P site with another tRNA in the A site. Directed by the bonding of an mRNA codon to the anticodon of a tRNA. Occurs in the ribosomes found in the cytoplasm of the cell.  TRANSCRIPTION DNA mRNA The Stages of Ribosome TRANSLATION Polypeptide Translation Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C A C G Anticodon A A A U G G U U U G G C 5’ Codons 3’ mRNA  1. Initiation Stage This stage brings together mRNA, the initiator tRNA, and the 2 subunits of the ribosome.  2. Elongation Stage Amino Acids are bonded 1 to another building the polypeptide chain out of the P site.  2. Elongation Stage Amino Acids are bonded 1 to another building the polypeptide chain out of the P site. 1 3 2  3. Termination Stage This stage is reached when the stop codon is recognized in the mRNA. There is NO tRNA that matches the stop codon.  TRANSCRIPTION DNA RNA 1 is transcribed from a DNA template. 3 Poly-A 5 RNA RNA transcript polymerase RNA PROCESSING Exon In2 eukaryotes, the RNA transcript RNA transcript (pre- (pre-mRNA) mRNA) is spliced and Intron modified to produce Aminoacyl-tRNA mRNA, which moves Cap synthetase from the nucleus to the NUCLEUS cytoplasm. Amino FORMATION OF acid INITIATION COMPLEX AMINO ACID ACTIVATION CYTOPLASM After leaving the tRNA 3 4Each amino acid nucleus, mRNA attaches attaches to its proper tRNA to the ribosome. with the help of a specific enzyme and ATP. mRNA Growing polypeptide Activated Poly-A amino acid Poly-A Ribosomal subunits Cap 5 TRANSLATION CC 5A succession of tRNAs A U add their amino acids to E A AC the polypeptide chain AA A Anticodon as the mRNA is moved UG G U U U A U G through the ribosome one codon at a time. Codon (When completed, the Ribosome polypeptide is released from the ribosome.)  Pt. 4 Mutations & Mutagens  Learning Objectives Describe various classes of mutations and their likely effects on gene expression Explain why different classes of mutagens cause different types of mutations  Mutations & Genes Remember… a gene is a sequence of DNA that encodes for a product, like a protein. A mutation is an change in the genetic material of the cell. typically referring to a change in the sequence of nitrogenous bases. Several Classes of Mutations Chromosomal Point Mutations Frameshift Mutations Mutations  Point Mutations Changes in a single base pair within the genome. When occurring in a gene, these mutations can affect the structure and function of the overall gene product (like a protein). 3 Categories of Point mutations Substitutions Insertions Deletions  Point Mutations – Substitutions A substitution point mutation – the replacement of 1 base pair with another. There are several different types of substitutions. Silent (Synonymous) The single base pair change does not cause an amino acid change in the polypeptide. Missense (Nonsynonymous) The single base pair change causes an amino acid change in the polypeptide. Can be very detrimental or not noticed at all. Nonsense The single base pair change causes a change from an amino acid to a stop codon. Usually very detrimental! Point Mutations – Insertions & Deletions A Insertion point mutation – the addition of 1 base pair within the genome. A Deletion point mutation – the removal of 1 base pair within the genome.  Frameshift Mutations These are changes in nucleotide base pairs within the genome that causes a reading frame shift. They occur in 2 possible ways. Insertions – adds new nucleotides in the genome Deletions – removes nucleotides from the genome GGG GCC GAC CGT ACC TG-  Chromosomal Mutations These are large scale nucleotide changes within a chromosome’s DNA. There are many categories of these mutations including: Insertions – addition of many nucleotides into the chromosome Deletions – removal of a section of the chromosome. Translocations - part of 1 chromosome is removed and inserted to somewhere in another chromosome. Inversions - reversal of a segment of the chromosome. Fusions - when 2 genes come together to form 1 new hybrid gene. (often caused by other mutations like insertions, deletions, and inversions) Duplications – copy of a region of the chromosome. Example – Genetic Mutations Original → The gray cat ran down the hall. Missense → The gray cat ran down the ball. Nonsense → The gray cat ran. Silent → The grey cat ran down the hall. Frameshift(In) → The grayish scat ran down the hall. Frameshift (del) → The gray cat ran he hall. Insertion → The gray green cat ran down the hall. Deletion → The gray down the hall. Duplication → The gray cat cat ran down the hall. Translocation → The gray down the hall cat ran. Inversion → The gray nar tac down the hall. Fusion → The gray cat ran downthe hall.  Mutagens vs. Spontaneous Changes Mutagens are physical or chemical agents that can cause mutations. Like radiation, X-rays, UV Arsenic, nitrosamine, nitrogen mustards (Bendamustine & Altretamine) Think about carcinogens! Spontaneous mutations Can occur during DNA replication, recombination, or repair