BIOL 1P91 - Chapter 12 STUDENT 2024 (1) PDF

Gene Expression at the Molecular Level I: Production of mRNA and Proteins Chapter 12 1  Chapter 12 Outline Overview of Gene Expression Transcription RNA Modification in Eukaryotes Translation and the Gen...

Gene Expression at the Molecular Level I: Production of mRNA and Proteins Chapter 12 1  Chapter 12 Outline Overview of Gene Expression Transcription RNA Modification in Eukaryotes Translation and the Genetic code The Machinery of Translation The Stages of Translation 2  Gene Expression & Mutation  Gene expression is the process by which the information of a gene is made into a functional product  Studied at both the molecular level & the trait level  A mutation is a heritable change in the genetic material  Can affect gene function by altering the gene sequence  Research focused on the effects of mutations proved instrumental in demonstrating the relationships between:  Normal genes and functional proteins  Mutated abnormal genes and non-functional proteins ClickOverview 12.1 to edit Master text of Gene Expression 3  Inborn Errors of Metabolism  Archibald Garrod studied patients with metabolic defects such as alkaptonuria  Inherited disease in which patient’s body accumulates abnormal levels of homogentisic acid (alkapton)  In 1908, Garrod proposed a relationship between inheritance of a mutant gene, a missing or defective enzyme, and a metabolic disease  Called this an inborn error of metabolism 4 Phenylalanin e metabolism 5  Beadle and Tatum  Discovered Garrod’s work in the early 1940s  Studied Neurospora crassa, common bread mold  Minimum requirements for growth are carbon source (sugar), inorganic salts, and biotin  Many different cellular enzymes are used to synthesize all molecules needed for growth from these components  Focused on amino acid synthesis  Hypothesized that genes encode enzymes, and a mutation might cause a defect in an enzyme needed for amino acid synthesis 6  Beadle and Tatum  Proposed that a different enzyme catalyzes each step in a biochemical pathway and that a different gene controls the production of each different enzyme  Collected different mutant strains that required supplementation with the amino acid arginine for growth  The pathway for arginine synthesis was thought to involve at least three precursor molecules  Therefore, three genes & three enzymes should be involved  Examined the ability of mutant strains to grow in the presence of the different precursor molecules 7  Beadle and Tatum  Mutant strains fell into three groups according to which enzyme was defective  Concluded that a single gene controls the synthesis of a single enzyme  The one gene – one enzyme hypothesis 8  Not Quite…  Enzymes are only one category of cellular proteins  Genes also encode for many other types of proteins  Some proteins are composed of two or more polypeptides  E.g. Hemoglobin is composed of two α-globin and two β-globin polypeptides  Some mRNAs can be spliced in alternative ways  Allows a single gene to encode more than one polypeptide  Some genes encode RNAs that are not used to make polypeptides 9 The Central Dogma of Gene Expression 10 Prokaryotes vs. Eukaryotes 11  Genes  A gene is an organized unit of DNA sequences that is transcribed into RNA and results in the formation of a functional product  Protein-coding genes are transcribed to produce a messenger RNA (mRNA) that specifies the amino acid sequence of a polypeptide  The polypeptide is considered to be the functional product  The mRNA is an intermediary in polypeptide synthesis  For non-coding RNA genes, the RNA itself is the final functional product  e.g. Transfer RNA (tRNA) and ribosomal RNA (rRNA) ClickTranscription 12.2 to edit Master text 12 Gene Organization 13 Three Stages of Transcription 14  A Closer Look at Elongation  RNA is synthesized in the 5’ to 3’ direction  Complementarity between ribonucleotides and DNA template guides sequence of RNA  Uracil substitutes for thymine  The strand of DNA that is used as a template for RNA synthesis is called the template or noncoding strand  The opposite DNA strand that is not used for transcription is called the coding strand  Has the same sequence as the mRNA (except T instead of U)  Unlike DNA polymerase:  RNA polymerases can start on a template without a primer  RNA polymerases do not proofread and have no exonuclease activity 15 Coding strand 16  Transcription along the chromosome  The DNA strand used as a template can vary for adjacent genes  Depends on position of promoter sequences  RNA is always synthesized 5’ to 3’  Template strand is read in the 3’ to 5’ direction 17  Eukaryotic Transcription  Basic features of transcription are identical between prokaryotic and eukaryotic organisms, but each step tends to involve a greater complexity of protein components  E.g. Eukaryotes have three RNA polymerases instead of one, and initiation of transcription requires five general transcription factors instead of sigma factor 18  Eukaryotic RNA Processing ClickRNA 12.3 to edit Master textin Eukaryotes Modification 19  RNA Processing: Capping  Covalent attachment of 7- methylguanosine to the 5’ end of the mRNA transcript  = 5’ cap  Occurs while RNA polymerase is still creating the pre-mRNA  5 cap is recognized by cap-binding proteins  Required for proper exit of the mRNA from the nucleus  Protects mRNA and helps it bind to a ribosome for translation 20  RNA Processing: Tailing  Poly adenylation sequence in mRNA attracts enzyme complex that cuts mRNA and adds 100 to 200 adenines to the 3’ end  = poly A tail  Aids in export from nucleus and increases mRNA stability  Allows mRNA to exist for a longer time in the cytosol 21  RNA Processing: Splicing  Many eukaryotic genes contain coding sequences that are interrupted by large segments of DNA that are transcribed but not translated  Exons are the RNA sequences found in the mature mRNA  Introns are intervening untranslated sequences  Splicing removes introns and joins exons together  Catalyzed by the spliceosome  Consists of several subunits (snRNPs) made of snRNA & protein  In alternative splicing, splicing can occur more than one way to produce different products 22 23  The Genetic Code  Specifies the relationship between the sequence of nucleotides in mRNA and the sequence of amino acids in a polypeptide  Sequence of an mRNA is read in groups of three consecutive ribonucleotide bases = codons  Specify for a particular amino acid, or indicate start or stop  e.g. CCC = proline, GGC = glycine  tRNAs bind to mRNA codons using anticodons ClickTranslation 12.4 to edit Master andtext the Genetic Code 24  The Genetic Code  Using 3 bases, 64 different codons are possible (= 43)  Only 20 different amino acids  Genetic code is degenerate  More than one codon can specify the same amino acid 25 Bacterial mRNA Organization 26  Reading Frame  The start codon defines the reading frame of an mRNA  Each adjacent codon is read as a triplet in the 5 to 3 direction  Insertion or deletion of any number of bases that is not a multiple of 3 changes the amino acid sequence 27 28  The Translation Machinery The Translation Machinery ClickThe 12.5 to edit Master text Machinery of Translation 29  tRNA  Different tRNAs are encoded by different genes  Common features  Cloverleaf structure  3’ single-stranded region for amino acid binding = Acceptor stem  Anticodon  Notation indicates amino acid  tRNAser carries serine  Aminoacyl-tRNA Synthetases  Enzymes that catalyze the attachment of amino acids to tRNA molecules (= Charging a tRNA)  Cells contain 20 different aminoacyl-tRNA synthetases  One for each of the 20 different amino acids  Two-step reaction results in a tRNA with an amino acid attached  = Charged tRNA or aminoacyl tRNA  Highly accurate process 31 32  Ribosomes  Macromolecular site where translation takes place  Composed of many different proteins & RNAs assembled into large and small subunits  Eukaryotic ribosomes consist of 40S and 60S subunits that combine to form an 80S ribosome  Prokaryotes have 30S and 50S subunits that form a 70S ribosome 33  RNA Ribosome Structure protein  Overall ribosome shape determined by rRNAs  tRNAs bind to the ribosome at three discrete sites:  A site = aminoacyl site  P site = peptidyl site  E site = exit site  Three Stages of Translation ClickThe 12.6 to edit Master Stages text of Translation 35 Translation Initiation in Bacteria 36  Eukaryotic Initiation Two key differences:  mRNAs do not contain a ribosomal binding site  Cap-binding proteins bind to 5’ cap, and promote binding of mRNA to small ribosomal subunit  Position of start codon is more variable  Small ribosomal subunit begins at 5’ cap and scans the mRNA  In many cases, first AUG codon is used as the start codon 37  Elongation – 1 1. Aminoacyl tRNA carrying a single amino acid binds to the A site  Via codon / anticodon recognition  Peptidyl tRNA (attached to polypeptide) is in the P site  Aminoacyl tRNA is in the A site 38  Elongation – 2 2. A peptide bond is formed between the amino acid at the A site and the polypeptide chain E P A  Catalyzed by rRNA  Ribosome is a ribozyme  Polypeptide transfers from tRNA in P site to amino acid in A site  = Peptidyl transfer reaction 39  Elongation – 3 3. Ribosome moves (translocates) toward the 3’ end of the mRNA by one codon  Shifts tRNAs down by one site E P A  Uncharged tRNA transfers from P site into E site, where it exits the ribosome  tRNA containing the polypeptide moves from A site into P site  The next codon is now exposed at an unoccupied A site 40  Termination  Translation ends when a stop codon is found in the A site  Recognized by proteins known as release factors  Binding of release factor to stop codon causes bond between polypeptide and tRNA in P site to break  Releases polypeptide and tRNA  Remaining subunits dissociate 41