From Gene to Protein Chapter 17

Overview of Transcription & Translation

One Gene - One Polypeptide Genes provide the instructions for making specific proteins Linear sequence of genes in DNA ultimately determines the linear sequence of amino acids in the polypeptide DNA does not make proteins directly Requires RNA

Transcription –Synthesis of RNA using DNA as a template –mRNA carries message from nucleus to cytoplasm Translation –Synthesis of a polypeptide under the direction of mRNA –Occurs in the cytoplasm

Structure of RNA Nucleotides –Ribose –Phosphate group –A, U, G, C Single stranded mRNA, tRNA, rRNA

Genetic Code DNA and RNA made of sequence of nucleotides (4 nucleotides) Polypeptides made of sequence of amino acids (20 amino acids) Code required to translate the sequence of nucleotides into a sequence of amino acids Triplet code of nucleotides as minimum code length to code for all 20 a.a. (4 3 =64)

Genetic Code Codon = three-nucleotide sequence in mRNA that specifies an amino acid 61 of 64 triplets code for amino acids (3 stop codons) Redundancy - more than 1 codon codes for each amino acid No ambiguity - A codon only codes for 1 amino acid

Read code in triplets from mRNA sequence

1. Initiation 2. Elongation 3. Termination

Promoter = region of DNA where RNA polymerase binds and where transcription begins –about 100 nucleotides long Transcription factors = DNA-binding proteins which bind to specific DNA nucleotide sequences at the promoter and help RNA polymerase recognize and bind the promoter –TATA box RNA polymerase unwinds the helix and begins transcription

Elongation RNA polymerase II –untwists the double helix, exposing bases at a time –adds RNA nucleotides to the 3’ end of the elongating strand –follows base-pairing rules: A-U, G-C mRNA grows about nucleotides/second Several molecules of RNA polymerase II can simultaneously transcribe the same gene –can produce proteins in large amounts

Termination of Transcription Transcription proceeds until RNA polymerase transcribes a terminator sequence which functions as a terminator signal –In prokaryotes, transcription stops right at end of termination signal –In eukaryotes, transcription proceeds about nucleotides past termination signal –In eukaryotes, most common termination signal is AAUAAA

Post-Transcriptional Modification 5’ cap = modified guanine nucleotide (GPPP) added to 5’ end of mRNA after transcription begins –protects the growing mRNA from degradation by hydrolytic enzymes –helps ribosome recognize attachment site for translation Poly-A tail = sequence of As added to 3’ end before it exits nucleus –Inhibit degradation of mRNA in cytoplasm, facilitate attachment of ribosome, facilitate export from nucleus to cytoplasm

RNA splicing removes introns and joins exons to form functional mRNA –Introns = intervening sequences = noncoding sequences of DNA; are transcribed, but not translated –Exons = expressed sequences = coding sequences of a gene RNA Splicing

Sliceosomes Enzymes excise introns and join exons to form a mRNA with a continuous coding sequence –Short nucleotide sequences at the end of introns are recognized by snRNPs which form a spliceosome which cuts introns at splice sites and splices exons together

Importance of Introns May play regulatory role in cell –May control gene activity –May help regulate the export of mRNA to the cytoplasm May allow a single gene to direct synthesis of different proteins –Single pre-mRNA is processed differently Increase probability of recombination to increase genetic diversity

Translation: Basic Concept Proteins are synthesized according to genetic message of sequential codons along mRNA –tRNA is the interpreter tRNA aligns appropriate a.a. to form new polypeptide by –transferring a.a. from cytoplasm’s a.a. pool to ribosome –recognizing the correct codons in mRNA Molecules of tRNA are specific for only one a.a.

Structure & Function of tRNA

Anticodon = nucleotide triplet in tRNA that base pairs with complementary codon in mRNA Wobble = ability of one tRNA to recongnize two or three different mRNA codons; occurs when the third base of the tRNA can H bond with more than one kind of base in the third position of the codon –U can pair with A or G –some tRNAs contain modified base (I) that can H bond with U, C, or A

Aminoacyl-tRNA Synthetase Catalyzes attachment of tRNA to a.a. 20 types of aminoacyl- tRNA synthetase –Active site fits only a specific combination of a.a. and tRNA Endergonic reaction driven by hydrolysis of ATP

Large and small subunits separated when not involved in protein synthesis About 60% rRNA and 40% protein mRNA binding site + 3 tRNA binding sites tRNA binding sites –A site = aminoacyl-tRNA binding site –P site = peptidyl-tRNA binding site –E site = exit site

Binding of small ribosomal subunit to mRNA and initiator tRNA –In prokaryotes, rRNA in small subunit base pairs with specific nucleotides in leader sequence of mRNA –In eukaryotes, the 5’ cap of the mRNA aids in the binding of leader sequence to small ribosomal subunit Start codon is AUG = methionine Initiation of Translation

Elongation Cycle of Translation peptidyl transferase Energy from hydrolysis of GTP

Termination of Translation Translation continues until termination codon (stop codon) is reached –Stop codons are UAA, UAG, UGA; do not code for amino acids Release factor binds to A site –Hydrolyzes bond between polypeptide and tRNA in P site –Polypeptide and tRNA released from ribosome –Translation complex dissociates

Polyribosomes Clusters of ribosomes that translate a mRNA at once, making many copies of the polypeptide Found in both prokaryotes and eukaryotes

Post-translational Modifications Primary structure (a.a. sequence) determines secondary and tertiary structure (3D) Chemical modification –Sugars, lipids, phosphate groups, … may be attached to some a.a. Chain-length modification –One or more a.a. may be cleaved from leading end of polypeptide chain –Single polypeptide chains may be divided into two or more pieces –Two or more polypeptides may join as subunits of a protein that has quaternary structure

Protein Destinations Signal peptides target some polypeptides to specific destinations in the cell Proteins made by free ribosomes function in cytosol Proteins made by bound ribosomes (attached to ER) generally destined for: –Membrane inclusion (membrane-bound enzymes) –Secretion from cell (hormones)

Polypeptides destined for endomembrane system or secretion marked by a signal peptide of hydrophobic a.a. at leading end of growing polypeptide Signal peptide recognized by SRP which binds signal peptide and then links to SRP receptor protein on ER membrane

Transcription & Translation in Prokaryotes One type of RNA polymerase No transcription factors No post- transcriptional modification Ribosomes smaller with different molecular composition Transcription and translation are simultaneous because no nucleus

Molecular Basis of Sickle-Cell Disease Point mutation = mutation in one to a few base pais in gene

Types of Point Mutations

Summary of Transcription & Translation