Molecular Biology DNA Expression Chapter 9 Part B

DNA Expression: The Central Dogma Genes contain specific sequences of bases coding the instructions for proteins In general one gene codes for one protein The basic process starts in the nucleus where enzymes transcribe the gene to make a strand of RNA. The RNA exits the nucleus through the nuclear pores. In the cytoplasm the RNA is translated into a sequence of amino acids (the building blocks of proteins) DNA RNA Protein Transcription Translation (Nucleus) (Cytoplasm)

Assembly of RNA on unwound regions of DNA molecule Transcription Assembly of RNA on unwound regions of DNA molecule mRNA rRNA tRNA mRNA processing proteins mature mRNA transcripts ribosomal subunits mature tRNA Convergence of RNAs Translation cytoplasmic pools of amino acids, ribosomal subunits, and tRNAs At an intact ribosome, synthesis of a polypeptide chain at the binding sites for mRNA and tRNAs Final protein

Transcription: from DNA to mRNA Transcription is the first step in gene expression Transcription occurs in the nucleus in eukaryotes The base sequence of a gene in DNA is copied to make a single strand of RNA

Transcription: from DNA to mRNA DNA is unwound Only the part of the chromosome containing the gene is unwound Makes a transcription bubble

Transcription: from DNA to mRNA RNA polymerase attaches to a binding site called a promoter The promoter is just in front of the gene (“up stream”)

Transcription: from DNA to mRNA RNA polymerase adds nucleotides one at a time using the DNA sequence as a template A-U, T-A, G-C, C-G

Transcription: from DNA to mRNA Transcription ends when the polymerase passes the end of the gene

Eukaryotic mRNA Processing Once RNA is transcribed it will be modified before leaving the nucleus for the cytoplasm Introns are spliced out Eukaryotic genes contain Exons which are coding regions Introns which are non-coding regions Before the RNA leaves the nucleus the introns are snipped out leaving just the exons

Eukaryotic mRNA Processing Once RNA is transcribed it will be modified before leaving the nucleus for the cytoplasm Protection from cytoplasmic RNA enzymes Poly A tail 50-300 adenines are added to the “tail” end (3’ end) 5’ “cap” A modified guanine is added to the “front” end (5’ end)

Translation Translation is the second step in gene expression Translation occurs in the cytoplasm The base sequence in the mRNA is decoded into a sequence of amino acids in a protein

Translation The Genetic Code The sequence of nucleotides/bases in the mRNA “spell” out the instructions for what protein the cell should make Different proteins determine the traits of cells and organisms Each set of three bases is one “word” or codon A codon indicates which amino acid to add to the protein Amino acids are protein monomers/subunits

Translation The Genetic Code There are 64 different codons 61 codons code for 20 amino acids Thus most amino acids are encoded by more than one codon 1 codon is used as a start signal AUG (codes for methionine) 3 codons are used as a stop signal UAA, UGA, UAG

Translation The Genetic Code 5’ ATG TCG GAC CTA 3’ 3’ TAC AGC CTG GAT 5’ 5’ AUG UCG GAC CUA 3’ (mRNA) met (start) ser asp leu

Translation tRNA Each tRNA has two attachment sites One is an anticodon A triplet of bases complimentary to mRNA codons The other attaches to the amino acid specified by the codon

Translation Initiation Translation is initiated when the initiator tRNA binds the first AUG (start codon) of the mRNA and a small ribosomal subunit, then a large ribosomal subunit joins them Initiator tRNA’s anticodon base pairs with AUG Amino acid is methionine (met) Initiator tRNA + mRNA + small and large ribosomal subunits = initiation complex

Translation Elongation Initiator tRNA+met lines up in the P site of the ribosome Anti-codon matches AUG Carries methionine Second tRNA+aa2 is placed in the A site of the ribosome The second tRNA’s anticodon matches the next three bases (codon) on the mRNA Carries the appropriate amino acid called aa2 to indicate it is the second amino acid Peptide bond forms between met and aa2 Initiator tRNA is released from the P site and from its amino acid, met

Translation Elongation The second tRNA+aa2-met complex moves to the P site of the ribosome Third tRNA+aa3 moves into the now vacant A site of the ribosome Peptide bond forms between aa2 and aa3 Forms a polypeptide consisting of three amino acids Met-aa2-aa3 This process continues adding an amino acid for each codon The polypeptide elongates

Translation Termination The ribosome encounters a stop codon in the mRNA There are no matching tRNAs for stop codons A release factor binds All the parts of the complex dissociate mRNA (can be used again) Small and large ribosomal subunits (can be used again) New polypeptide chain The new polypeptide will either stay in the cytoplasm or enter the rough endoplasmic reticulum of the endomembrane system

Mutations Mutations are changes in the nucleotide sequence of DNA These can affect the sequence of amino acids in the encoded polypeptide thus affecting the function of the protein

Mutations Common gene mutations Base-pair substitution A single base pair changes Affects one codon and thus one amino acid

Mutations Common gene mutations Insertions One or more base pairs are added to the original DNA sequence Results in a reading frame shift

Mutations Common gene mutations Deletions One or more base pairs are removed from the original DNA sequence Results in a reading frame shift

Mutations Common gene mutations Frameshift (due to insertion or deletion) Shifts the 3-bases-at-a-time reading frame Changes the amino acid sequence from the point of mutation on. The cat ate the rat Tec ata tet her at (deletion) Thr eca tat eth era t (insertion)

Mutations Mutation example: Sickle-cell anemia In the DNA sequence coding for the beta chain in hemoglobin there is a single base-pair substitution Hemoglobin carries oxygen through the blood When that DNA is transcribed to mRNA the mistake is copied When the mRNA is translated into a polypeptide one different amino acid is used Normal sequence: val-his-leu-thr-pro-glu-glu- Mutated sequence: val-his-leu-thr-pro-val-glu- This one change disrupts the normal 3D folding of the protein Mis-shaped hemoglobin causes red blood cells to distort into a sickle shape under low oxygen conditions Causes clotting and disrupts blood circulation

Summary DNA expression Transcription Translation Gene Mutations Genes “code” for proteins and thus traits Transcription Translation Genetic code Gene Mutations