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Ch. 17: From Gene to Protein The Connection Between Genes and Proteins • The study of metabolic defects provided evidence that genes specify proteins – Garrod, suggested phenotypes had to do with expression of genes for enzymes • Transcription and translation are the two main processes linking gene to protein – Copy the information and interpret the information • In the genetic code, nucleotide triplets specify amino acids – Sequence of nucleotides = primary protein structure • The genetic code must have evolved very early in the history of life – DNA code is universal…..all cells use the same codons and amino acids to make their various proteins Back to Mendel • One of Mendels “factors” for peas was stem length. We say “height” and the alleles are tall and short. Actually it’s “length” and the stems are long or not-long • Normal peas have a gene for the hormone called gibberellin which stimulates stem elongation. • Dwarf peas lack this gene and do not make gibberellin and are therefore not tall. • Dwarf peas will grow to normal height if gibberellins are added to their water • PROTEINS ARE THE LINKS BETWEEN GENOTYPE AND PHENOTYPE. Scientific Evidence • 1909 – “inborn errors of metabolism” – alkaptonuria • 1930 – Beadle and Ephrussi, eye color in flies is due to an enzyme for pigment production • Beadle and Tatum – minimal medium Neurospora crassa (bread mold), used x-rays to create mutations, complete media had 20 amino acids, looking for inability to metabolize amino acids from a limited source – Mutants had defects in metabolism – Must be enzymes related – Enzymes are proteins – One gene – one enzyme hypothesis – now modified to one gene – one (protein) polypeptide Beadle and Tatum’s Neurospora crassa Experiment Transcription and Translation • Genes have instructions for making proteins, but genes do not make proteins directly • Transcription is the synthesis of RNA under the direction of DNA. DNA provides the template. Get an accurate copy; mRNA • Translation is the actual synthesis of a polypeptide, at the ribosome, under the direction of the mRNA • DNA RNA protein (polypeptide) Terminology • Triplet code: three DNA nucleotides = a word • mRNA: carries message from DNA to ribsome • tRNA: transports amino acids within cytoplasm rRNA: ribosomes are composed of rRNA and proteins • Ribosome: solid organelle found in cytoplasm of ALL cells; used to manufacture protein • Template strand: for each gene only one side of the DNA is transcribed • Codon: mRNA triplets are called codons • Reading frame: 5’3’, starting at beginning, groups of three – The red dog ate the cat – xHer edd oga tat hec atx Cracking the Code • 1960 – Marshall Nirenberg at NIH – National Institute of Health – www.nih.gov – Human genome projects library • Translated all the possible codons into amino acids • Found codons for “start” and for “stop” • Several amino acids can be coded for with more than one codon (redundancy) but no codons are for multiple amino acids (no ambiguity) • “wobble effect” Code Evolved Early in the History of Life on Earth (and any life anywhere else too) • Code is (near) universal to all know/studied organisms…. Bacteria can translate human genetic information • All modern organisms have a common ancestor • Few exceptions are found in protista and in mitochondria DNA (…. Endosymbiont hypothesis….) • Sketch a DNA molecule with chemically correct details • Show how it would replicate and • How it would transcribe and • List the amino acids in the short polypeptide it forms • It has the following template strand sequence of DNA triplets – TAC TTT GAG ATT Genomic like information • Stthegeneticcodeisnearlyuniversalpsharedbyorg anismsfromthesimplestbacteriatothemostcomple xplantsandanimalspstthernacodonccgpforinstan cepistranslatedastheaminoacidprolineinallorgani smswhosegeneticcodehasbeenexaminedpstinla boratoryexperientspgenescanbetranscribedandtr anslatedaftertheyaretransplantedfromonespecie stoanotherpfponeimportantapplicationisthatbacte riacanbeprogrammedbytheinsertionofhumangen estosynthesizecertainhumanproteinsthathaveim portantmedicalusesp The Synthesis and Processing of RNA • Transcription is the DNA directed synthesis of RNA • Eukaryotic cells modify RNA after transcription Transcription • RNA polymerase fits onto DNA (3’) and moves in a 5’ 3’ direction for the synthesis of the RNA strand. • C with G and this time, A with U (uracil) • DNA acts as a template • DNA is only opened at a small region (gene or genes of interest) • DNA helix reseals as RNA polymerase passes by…. Completely intact and undiluted. Bacterial transcription • Eukaryotic cells have 3 kinds of RNA polymerase (I, II – used in RNA synthesis and III) • Bacteria have one kind – it makes not only mRNA but also other types of RNA • Bacteria have one chromosome and many plasmids. Information is constantly being sent to ribosomes for translation into proteins needed by the bacterial cell Steps of Transcription 1. Initiation Promoter – region where polymerase attaches and a dozen bases upstream; start here and use this side of the helix. Collection of transcription factors initiate the “complex” – TATA box 2. 3. Elongation DNA exposed 20 bases at a time 5’ 3’ synthesis of RNA strand RNA peels away from DNA as completed rate is 60 nucleotides per second Termination DNA contains a terminator sequence polymerase continues to a AAUAAA sequence and 10-35 nucleotides later the preRNA is cut free other details are still ‘murky’ Modification of RNA • Initially RNA is called preRNA • The 5’ end (transcribed 1st) is capped with special guanine – provides protection and a start here signal for translation • Other end gets a ploy A tail (AAA-AAA) – in addition to ribosomal attachment and protection, it seems to facilitate RNA as it leaves the nucleus • These regions are nontranslated Further modification of RNA • Most of the pre RNA is actually removed…. It didn’t code for information about how to make a protein. We are uncertain of the function of this info, which does not make the info unimportant. • Initially the RNA can be 8000 bases, actual info for protein that goes to ribosomes is about 1200 or 400 amino acids (1200 bases/ 3 bases per codon) “Cut and Paste” • Called RNA splicing • Introns (intervening segments) are removed – they are noncoding, short, repetitive sequences, unique – cause restriction enzymes to cut segments differently and create the DNA fingerprint – Probably have a role in gene expression and activity – May be place where new proteins evolve – Increase odds of crossing over during synapsis of tetrads (meiosis II) • Exons (expressed) – these are translated into amino acids for the polypeptide – 150 nucleotides • 5’ cap + exon + exon + exon + …. + poly A tail • Process requires snRNP’s - small nucleotide ribonucleoproteins…. Sites to bind • Ribozymes = RNA that functions as an enzyme.