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Chapter 6 From DNA to Protein: How Cell Read the Genome Genetic information directs the synthesis of protein The central dogma of molecular biology Genes can be expressed with different efficiencies Untranscribed portions Nucleotide The chemical structure of RNA differs slightly from that of DNA 3’ phosphodiester bond 5’ Uracil forms a base pair with adenine 2 hydrogen bonds RNA molecules can form intramolecular base pairs and fold into specific structures Base-pair with complementary sequences Conventional base-pair interactions Nonconventional base-pair interactions Transcription produces an RNA complementary to one strand of DNA Coding strand Sense strand Non-Coding strand Anti-sense strand Template strand Sense & antisense strand DNA duplication by DNA polymerase Transcription by RNA polymerase DNA is transcribed by the enzyme RNA polymerase ATP CTP UTP GTP Transcription can be visualized in the electron microscope rRNAs Gene 1 RNA polymerase Gene 2 DNA Ribosomal proteins RNA polymerase vs DNA polymerase RNA polymerase DNA polymerase Ribonucleotides Deoxyribonucleotides Without primer With primer 1/104 error rate 1/107 error rate Types of RNA produced in cells messenger RNA non-messenger RNA ribosomal RNA microRNA transfer RNA Signals in the sequence of a gene tell bacteria RNA polymerase where to start and stop transcription Bacterial RNA polymerase Chain elongation Bacterial promoters and terminators have specific nucleotide sequences that are recognized by RNA polymerase Bacterial RNA polymerase Some genes are transcribed using one strand DNA as a template, whereas others are transcribed using the other DNA strand The direction of transcription is determined by the orientation of the promoter at the beginning of each gene The three RNA polymerases in eucaryotic cells mRNA sRNAs sRNAs To begin transcription, eucaryotic RNA polymerase II requires a set of general transcriptional factors -25 TATA-binding protein Dramatic local distortion in the DNA Phosphorylate the tail of RNA polymerase II Allow the template strand to be exposed by ATP hydrolysis Dephosphorylated form Transcription initiation complex TATA-binding protein (TBP) binds to TATA box sequences and distorts the DNA TBP (TATA-binding protein) TATA box DNA Before they can be translated, mRNA molecules made in the nucleus move out into the cytoplasm via pore in the nuclear envelope Pores in nuclear envelope TEM Phosphorylation of RNA polymerase II allows RNA-processing proteins to assemble on its tail TFIIH (1) Capping (2) Polyadenylation (3) Splicing Eucaryotic mRNA molecules are modified by capping and polyadenylation (1) Start after 25 nucleotides has been polymeized (1) To increase the stability of the eucaryotic mRNA molecule Functions:(2) To aid its export from the nucleus to the cytoplasm (3) To identify the RNA molecule as an mRNA Eucaryotic mRNA molecules are modified by capping and polyadenylation (2) CH3 2 1 3 4 5 Eucaryotic and bacterial genes are organized differently Promoter Intron is longer than exon Most human genes are broken into exons and introns 3 exons 26 exons Special nucleotide sequences signal the beginning and the end of an intron Branch point of the lariat long intron-exon boundary (border) short R: A or G Y: C or U N: A or C or G or U RNA splicing might occurred before or after polyadenylation RNA splicing An introns forms a branched structure during splicing 5’ Branch point of the lariat 3’ 5’ Spliceosome Small nuclear RNAs (snRNAs) + Proteins = Small nuclear ribonucleoprotein particles (snRNPs) The -tropomyosin gene can be spliced in different ways (1) Many different protein to be produced from the same gene by alternative splicing (2) 60% of human genes probably undergo alternative splicing A specialized set of RNA-binding proteins signal that a mature mRNA is ready for export to the cytoplasm Recognizes and exports only completed mRNAs Exon junction complex (EJC) Mark completed RNA splices Life times depends on (1) Nucleotide sequence (3’ untranslated sequence) (2) The type of cell 3’ untranslated region Procaryote and eucaryotes handle their RNA transcripts differently Transcription in procaryotic or eucaryotic cells (1) Transcription in procaryotic or eucaryotic cells (2) Bacteria Eucaryotic cells RNA polymerase Single type Three types (I, II, III) Accessory proteins X General transcription factors Nontranscribed DNA between genes Short Long Small RNAs (1) siRNA (small interfering RNA) (2) miRNA (microRNA) (3) piRNA (piwi-interacting RNA) miRNA siRNA Amino Three-nucleotide codons acids The nucleotide sequence of an mRNA is translated into the amino acid sequence of a protein via the genetic code Start codon Stop codons UUU codes for phenylalanine Marshall Nirenberg Heinrich Matthaei Messages of mixed repeating sequences further narrowed the coding possibilities Gobind Khorana The Nobel Prize in Physiology or Medicine 1968 Interpretation of the genetic code and its function in protein synthesis An RNA molecule can be translated in three possible reading frames Reading frame Frame shift tRNA molecules are molecular adaptors, linking amino acids to codons L-shape molecule dihydrouridine pseudouridine Wobble base-pairing 61 codons 31 anticodons 20 amino acids The genetic code is translated by means of two adaptors that act one after another Charging 1 Aminoacyl-tRNA synthase 2 Charged tRNA Anticodon Ribosomes are found in the cytoplasm of a eucaryotic cell Attached to the ER Ribosome Free in the cytosol TEM A ribosome is a large complex of four RNAs and more than 80 proteins (rRNA) (rRNA) Catalyzes the formation of polypeptide chain Matches the tRNA to the codon of the mRNA 1/3 2/3 Each ribosome has a binding site for mRNA and three binding sites for tRNA Large subunit peptidyl-tRNA Large subunit Small subunit Exit aminoacyl-tRNA Small subunit Translation takes place in a four-step cycle Catalyzed by an enzymatic site in the large subunit Large subunit Ribosomal RNAs give the ribosome its overall shape Rate of sedimentation in an ultracentrifuge Protein Catalytic site for peptide bond formation Large subunit of a bacterial ribosome Initiation of protein synthesis in eucaryotes requires initiation factors and a special initiator tRNA or formylmethionine in bacteria only this charged RNA can binds to the P-site E E P P A A Start codon E P A In the final phase of protein synthesis, the binding of release factor to an A-site bearing a stop codon terminates translation Stop codons UAG UGA UAA A single procaryotic mRNA molecule can encode several different proteins Operons Polycistronic Procaryote Proteins are translated by polyribosomes Polyribosomes (Polysomes) Inhibitors of procaryotic protein synthesis are used as antibiotics The proteosome degrades short-lived and misfolded proteins Active site of the proteases Ubiquination for protein degradation Ubiquitin Protein production in a eucaryotic cell requires many steps Many proteins require additional modification to become fully functional Glycosylation (> 100 kinds) An RNA world may have existed before modern cells arose An RNA molecule can in principle guide the formation of an exact copy of itself Ribozyme Ribozyme – RNA molecules that possess catalytic activity A ribozyme is an RNA molecule that possesses catalytic activity Biochemical reactions that can be catalyzed by ribozymes Could an RNA molecule catalyze its own synthesis? RNA may have preceded DNA and proteins in evolution