Unformatted text preview: Transcrip)on • Informa)on is stored in the sequence of nucleo)des • DNA inherited by an organism leads to speciﬁc traits – Proteins link genotype and phenotype Gene expression • The process by which DNA directs protein synthesis • Gene expression includes two stages: – Transcrip)on – Transla)on • Central dogma of molecular biology Genes specify proteins via transcrip)on and transla)on • How was the fundamental rela)onship between genes and proteins discovered? – Observa)on – Symptoms of a disease Nutri&onal Mutants • Expose bread mold to X-‐rays • Mutants unable to survive on minimal media Growth: Wild-‐type cells growing and dividing No growth: Mutant cells cannot grow and divide Minimal medium • Crosses iden)ﬁed diﬀerent classes of arginine-‐
deﬁcient mutants RESULTS Classes of Neurospora crassa Wild type CondiGon Minimal medium (MM) (control) MM + ornithine MM + citrulline MM + arginine (control) Class I mutants Class II mutants Class III mutants One gene – one enzyme hypothesis CONCLUSION Gene A Gene B Gene C Wild type Class I mutants (mutaGon in gene A) Class II mutants (mutaGon in gene B) Class III mutants (mutaGon in gene C) Precursor Precursor Precursor Precursor Enzyme A Ornithine Enzyme B Citrulline Enzyme A Ornithine Enzyme B Citrulline Enzyme A Ornithine Enzyme B Citrulline Enzyme A Ornithine Enzyme B Citrulline Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine • Some proteins aren’t enzymes: • Many proteins are composed of several polypep)des, each of which has its own gene Beadle and Tatum Summary • One gene -‐ one enzyme • One gene -‐ one protein • One gene -‐ one polypep)de • One gene -‐ one RNA Basic Principles of Transcrip)on and Transla)on • TranscripGon – Under the direc)on of DNA – mRNA • TranslaGon – Under the direc)on of mRNA – Ribosomes DNA TRANSCRIPTION mRNA Ribosome TRANSLATION • Prokaryotes – No addi)onal processing PolypepGde Bacterial cell • Eukaryotes – Nuclear envelope – RNA processing Nuclear envelope DNA TRANSCRIPTION Pre-‐mRNA RNA PROCESSING mRNA TRANSLATION Ribosome PolypepGde EukaryoGc cell The Gene)c Code • Triplet code Coding Problem • If one nucleo)de codes for one amino acid, how many diﬀerent amino acids can be speciﬁed? • How about two nucleo)des? • How about three nucleo)des? Transcrip)on DNA molecule Gene 2 Gene 1 Gene 3 DNA template strand TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid Transla)on • Codons • Each codon speciﬁes an amino acids DNA molecule Gene 2 Gene 1 Gene 3 DNA template strand TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid The Gene)c Code Third mRNA base (3ʹ′ end of codon) First mRNA base (5ʹ′ end of codon) • 64 triplets • 61 code for amino acids • 3 are “stop” codons • One start codon • Redundant but not ambiguous • Universal Second mRNA base Transcrip)on is the DNA-‐directed synthesis of RNA • Follows base-‐pairing rules as DNA • Why do we need RNA? RNA Polymerase Nontemplate strand of DNA ElongaGon RNA polymerase 3ʹ′ RNA nucleoGdes 3ʹ′ end 5ʹ′ 5ʹ′ DirecGon of transcripGon ( downstream ) Newly made RNA Template strand of DNA • Promoter • Transcrip)on Unit • Terminator Synthesis of an RNA Transcript Three stages of transcrip)on: • Ini)a)on • Elonga)on • Termina)on 1 Promoter 5ʹ′ 3ʹ′ TATA box TranscripGon factors Template Start point 3ʹ′ 5ʹ′ Template DNA strand 2 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ 3 . RNA polymerase II TranscripGon factors 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ 5ʹ′ RNA transcript TranscripGon iniGaGon complex Ini)a)on of Transcrip)on – Promoter – TATA Box – Transcrip)on factors – Transcrip)on ini)a)on complex Promoter TranscripGon unit 5ʹ′ 3ʹ′ Start point RNA polymerase 3ʹ′ 5ʹ′ DNA 1 IniGaGon 5ʹ′ 3ʹ′ Unwound DNA RNA transcript Template strand of DNA 3ʹ′ 5ʹ′ 2 ElongaGon Rewound DNA 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ RNA transcript 3ʹ′ 5ʹ′ • Elonga)on – Transcrip)on progresses at a rate of 40 nucleo)des per second in eukaryotes • Termina)on – Bacteria – Eukaryotes RNA Processing: Eukaryo)c cells modify RNA a\er transcrip)on • Enzymes modify pre-‐mRNA • Both ends of the primary transcript are altered • Some interior parts of the molecule are cut out Each end of a pre-‐mRNA molecule is modiﬁed 5ʹ′ Protein-‐coding segment PolyadenylaGon signal G P P P 5ʹ′ Cap AAUAAA 5ʹ′ UTR Start codon Stop codon • 5ʹ′ cap – Methyla)on of 5’ G • 3ʹ′ end poly-‐A tail – Polyadenyla)on Func)ons of modiﬁca)ons : – Facilitate export – Protect mRNA – Help ribosomes a^ach 3ʹ′ UTR 3ʹ′ AAA … AAA Poly-‐A tail RNA Splicing • Exons • Introns • RNA splicing removes introns and joins exons – 5ʹ′ Exon Intron Pre-‐mRNA 5ʹ′ Cap Exon Exon Intron 3ʹ′ Poly-‐A tail 1 30 31 Coding segment mRNA 5ʹ′ Cap 1 5ʹ′ UTR 104 146 105 Introns cut out and exons spliced together Poly-‐A tail 146 3ʹ′ UTR 5ʹ′ 5ʹ′ RNA RNA ttranscript ranscript ((pre-‐mRNA) pre-‐mRNA) Exon Exon 11 Intron Intron Protein Protein snRNA snRNA Exon Exon 22 Other Other proteins proteins snRNPs snRNPs Spliceosome • Spliceosomes consist of: 5ʹ′ – Proteins – snRNPs Spliceosome components 5ʹ′ mRNA Exon 1 Exon 2 Cut-‐out intron Ribozymes • Three proper)es of RNA enable it to func)on as an enzyme – It can form a 3-‐D structure – Contain func)onal groups – May hydrogen-‐bond Alterna)ve RNA Splicing • Some genes can encode more than one kind of polypep)de – Dependent on which segments are treated as exons during RNA splicing – The number of diﬀerent proteins an organism can produce is much greater than its number of genes Proteins o\en have a modular architecture consis)ng of discrete regions called domains DNA Gene Exon 1 Intron Exon 2 Intron Exon 3 TranscripGon RNA processing TranslaGon Domain 3 Domain 2 Domain 1 PolypepGde Transla)on is the RNA-‐directed synthesis of a polypep)de Amino acids PolypepGde Ribosome Phe tRNA with amino acid a[ached Gly tRNA AnGcodon Codons 5ʹ′ mRNA 3ʹ′ A cell translates mRNA into protein with the help of transfer RNA Amino acids PolypepGde Molecules of tRNA are not iden)cal: – Speciﬁc amino acid – An)codon Ribosome Phe tRNA with amino acid a[ached Gly tRNA AnGcodon Codons 5ʹ′ mRNA 3ʹ′ Transfer RNA • A tRNA molecule consists of a single RNA strand about 80 nucleo)des long 3ʹ′ Amino acid a[achment site 5ʹ′ Hydrogen bonds AnGcodon tRNA twists and folds into a L-‐shape 5ʹ′ • 3ʹ′ Amino acid a[achment site Hydrogen bonds 5ʹ′ 3ʹ′ AnGcodon AnGcodon Symbol used in this book Aminoacyl-‐tRNA synthetase (enzyme) Amino acid Accurate transla)on requires: 1. Correct match by enzyme aminoacyl-‐
tRNA synthetase 2. Correct match between an)codon and codon P P P Adenosine ATP P Adenosine P P i P i P i tRNA Aminoacyl-‐tRNA synthetase tRNA P Adenosine AMP Computer model Aminoacyl-‐tRNA ( charged tRNA ) Ribosomes facilitate speciﬁc coupling of tRNA an)codons with mRNA codons • Two ribosomal subunits • Subunits made of proteins and ribosomal RNA tRNA molecules Growing polypepGde Exit tunnel E P A Large subunit Small subunit 5ʹ′ mRNA 3ʹ′ Func)ons of Ribosome • Hold mRNA and tRNA • Catalyze joining of amino acids to the growing pep)de chain A ribosome has three binding sites for tRNA: P site (PepGdyl-‐tRNA binding site) E site (Exit site) mRNA binding site • P site • A site • E site A site (Aminoacyl-‐ tRNA binding site) E P A Large subunit Small subunit Building a Polypep)de The three stages of transla)on: • Ini)a)on • Elonga)on • Termina)on Ini)a)on of Transla)on • Brings together mRNA, a tRNA, and the ribosome 3ʹ′ U A C 5ʹ′ Met 5ʹ′ A U G 3ʹ′ IniGator tRNA mRNA 5ʹ′ P site GTP Start codon mRNA binding site 3ʹ′ Small ribosomal subunit Me t Large ribosomal subunit GDP E 5ʹ′ A 3ʹ′ TranslaGon iniGaGon complex Elonga)on of the Polypep)de Chain Amino end of polypepGde Ribosome ready for next aminoacyl tRNA E 3ʹ′ mRNA 5ʹ′ P A site site GTP GDP E E P A P A GDP GTP E P A Termina)on of Transla)on • Occurs when a stop codon reaches the ribosome • A site accepts a protein called a release factor Release factor Free polypepGde 5ʹ′ 3ʹ′ 5ʹ′ Stop codon (UAG, UAA, or UGA) 5ʹ′ 3ʹ′ 2 GTP 2 GDP 3ʹ′ Polyribosomes enable a cell to make many copies of a polypep)de very quickly • Polyribosome (or polysome) Completed polypepGde Growing polypepGdes Incoming ribosomal subunits Start of mRNA (5ʹ′ end) Ribosomes Polyribosome mRNA End of mRNA (3ʹ′ end) Comple)ng the Func)onal Protein • Post Transla)onal modiﬁca)on – Ac)va)on by enzymes – Mul)ple subunits come togeth • Protein folding • Completed proteins are targeted Targe)ng Polypep)des to Speciﬁc Loca)ons • Popula)ons of ribosomes: – Free ribosomes – Bound ribosomes • Ribosomes are iden)cal Targe)ng Polypep)des to Speciﬁc Loca)ons Ribosome mRNA Signal pepGde Signal pepGde removed Signal-‐ recogniGon parGcle (SRP) CYTOSOL ER LUMEN SRP receptor protein TranslocaGon complex ER membrane Protein Muta)ons can aﬀect protein structure and func)on • Point mutaGons Wild-‐type hemoglobin DNA Mutant hemoglobin DNA C T T C A T 3ʹ′ 5ʹ′ 5ʹ′ 3ʹ′ 5ʹ′ G T A G A A 3ʹ′ 5ʹ′ 3ʹ′ mRNA G A A 5ʹ′ mRNA G U A 3ʹ′ 5ʹ′ Normal hemoglobin Glu 3ʹ′ Sickle-‐cell hemoglobin Val Types of Muta)ons • Base-‐pair subs)tu)ons – Missense muta)ons – Silent muta)on – Nonsense muta)on • Base-‐pair inser)ons or dele)ons – Frameshi\ muta)on Wild type DNA template 3ʹ′ strand 5ʹ′ 5ʹ′ 3ʹ′ mRNA 5ʹ′ 3ʹ′ Protein Stop Amino end Carboxyl end A instead of G 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ U instead of C 5ʹ′ 3ʹ′ Stop Wild type DNA template 3ʹ′ strand 5ʹ′ 5ʹ′ 3ʹ′ mRNA 5ʹ′ 3ʹ′ Protein Stop Amino end Carboxyl end T instead of C 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ A instead of G 3ʹ′ 5ʹ′ Stop Wild type DNA template 3ʹ′ strand 5ʹ′ 5ʹ′ 3ʹ′ mRNA 5ʹ′ 3ʹ′ Protein Stop Amino end Carboxyl end A instead of T 3ʹ′ 5ʹ′ 5ʹ′ 3ʹ′ U instead of A 5ʹ′ 3ʹ′ Stop Wild type DNA template 3ʹ′ strand 5ʹ′ 5ʹ′ 3ʹ′ mRNA 5ʹ′ 3ʹ′ Protein Stop Amino end Carboxyl end Extra A 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ Extra U 5ʹ′ 3ʹ′ Stop Wild type DNA template 3ʹ′ strand 5ʹ′ 5ʹ′ 3ʹ′ mRNA 5ʹ′ 3ʹ′ Protein Stop Amino end Carboxyl end missing 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ missing 5ʹ′ 3ʹ′ Wild type DNA template 3ʹ′ strand 5ʹ′ 5ʹ′ 3ʹ′ mRNA 5ʹ′ 3ʹ′ Protein Stop Amino end Carboxyl end missing 5ʹ′ 3ʹ′ 3ʹ′ 5ʹ′ missing 5ʹ′ 3ʹ′ Stop Occurrence of muta)ons • Spontaneous muta)ons • Mutagens ...
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