Genetics Exam 3 Lecture Objectives Genetic Code Transcription Chapter 12 Transcription Process by which RNA molecules are synthesized on a DNA template Results in an mRNA molecule complementary to the gene sequence on one of the double helix s 2 strands catalyzed by RNA polymerase which uses Uracil instead of thymine n NTP DNA RNA Pol enzyme NMP n n PPi Studies by Nirenberg and Matthaei deciphered first specific coding sequences use of in vitro cell free protein synthesizing system and an enzyme polynucleotide phosphorylase PNP polynucleotide phosphorylase allowed artificial synthesis of RNA templates which could be added to the cell free system PNP doesn t require a DNA template and probability of insertion into growing RNA strand depends on the concentration of available ribonucleotides Nirenberg and Matthaei synthesized RNA homopolymers e g AAAAAA UUUUUU CCCCCC or GGGGGG and tested each mRNA to see what amino acids would be incorporated Radioactive labeling RNA heteropolymers experiments used 2 or more different ribonucleoside diphosphates added in combination to form different messages The frequency of the amino acids that are synthesized depends on the relative concentration of one ribonucleotide to another Allowed researchers to determine composition of code words but not sequence Base transition mutation where a purine is switched for another purine A for G or a pyrimidine is switched for another Pyrimidine C for T or U 5 10X more common than transversions Base transversion mutation where a purine is switched for a pyrimidine or vice versa Purines Pyrimidines Adenine and Guanine Cytosine Thymine Uracil The genetic code coding dictionary reveals function of 64 triplets The genetic code is degenerate one a a can be coded by more than 1 triplet The genetic code is unambigous single triplet specifies only 1 a a Ordered Code Chemically similar amino acids a a often share 1 or 2 middle bases e g U or C present in 2nd position of triplets for hydrophobic a a such as Valine or Alanine AAA and AAG are positively charged Lysine but AGA and AGG are positively End result of an ordered code is that it buffers the potential effect of mutation on protein charged Arginine function Wobble hypothesis Crick 3rd base in triplet has more flexibility allows anticodon of a single form of tRNA to pair with more than 1 triplet mRNA Start Codon AUG codes for Methionine Requirements for RNA polymerase Template is 1 strand of the DNA double helix uses Nucleoside triphosphates NTPs as substrates A C G U synthesizes in 5 3 direction No primer needed Holoenzyme is active form of the enzyme Sigma factor plays regulatory role in initiation of RNA transcription many types Promoters Initial step of transcription is template binding where RNA pol subunit recognizes a specific DNA sequence called a promoter located upstream 5 from point of initial transcription of gene promoters govern the efficiency of initiation of transcription Consensus sequences of DNA are conserved throughout evolution and are found in bacterial promoters TATAAT is 10 nucleotides upstream from initial transcription TTGACA is 35 nucleotides upstream from initial transcription Eukaryotic Promoters TATA box 10 nucleotides upstream Post transcriptional mRNA processing 7 methylguanosine cap 7 mG added to 5 end of transcript added before initial transcript is complete protects 5 end from nuclease degradation may be involved in transport of mature mRNAs across nuclear membrane into added to 3 end of initial transcript after it is cleaved enzymatically at a position of 10 35 ribonucleotides from a highly conserved AAUAA sequence detected in nearly all euk and some prok mRNA without tail is rapidly degraded critical to transport out of nucleus into cytoplams Introns intervening are non coding regions of the mRNA transcript Exons expressed are the regions retained and expressed in final translation cytoplasm poly A tail Splicing product Splicing Spliceosomes catalyze the splicing of introns and exons small nuclear RNAs snRNAs are complexed with proteins to form small nuclear ribonucleoproteins snRNPS snurps are involved in the splicing introns are cut out and exons are joined specific bases are found at the intron exon borders spliceosomes recognize the sequence at the intron exon borders and branch point pulls the exons together and promotes rearrangement of the phosphodiester bonds intron cut out and exon joined Drawing of a monocistronic one protein product gene Translation and Proteins Chapter 13 Major components of translation mRNA message consisting of ribonucleotides A U C G that contains information about the amino acids used to build a protein Ribosomes tRNA transfer RNA complex of proteins and RNAs rRNAs that builds the protein Prok s have 70S ribosome complex with 50S large subunit and 30S small subunit Euk s have 80S ribosome complex with 60S large and 40S small bring amino acids to the ribosome structure is a 2D cloverleaf secondary structure based on hydrogen bonding anticodon loop contains the anticodon that matches a specific codon that codes for an a a 3 acceptor region is where a a is bound tRNA charging reaction tRNA molecules must be linked to respective a a called charging occurs by enzymes called aminoacyl tRNA synthetases 20 different enzymes corresponding to 20 a a Initial step Step 2 a a is converted to active form by reacting with ATP to form an aminoacyladenylic acid Covalent linkage is formed b w 5 phosphate group of ATP and the carboxyl end of the a a reacts with specific tRNA molecule a a is transferred to appropriate tRNA and bonded covalently to the adenine residue at the 3 end now charged may participate directly in protein synthesis Aminoacyl tRNA synthetases are highly specific enzymes because they only recognize 1 a a and the subset of corresponding tRNAs called isoaccepting tRNAs Translation tRNA charging described above 1 2 Chain Initiation a initiation involves the small ribosomal subunit an mRNA molecule a specific charged tRNA GTP Mg2 and 3 initiation factors IF b AUG start codon calls for modified a a formylmethionine fmet c Step 1 Small ribosomal subunit binds to IF1 and this complex binds to mRNA IF2 and IF3 bind to complex initiator tRNA fmet binds to mRNA codon in the P site this represents the initiation complex GTP hydrolyzed providing energy for release of IF factors EF Tu binds to tRNA facilitating entry into A site a P peptidyl site and A aminoacyl site and E exit site b Step 1 next charged
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