Lecture 12a: The Genetic Code PDF

Lecture 12a 12a/ The Genetic code Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 27: p1005-1014 PROTEIN METABOLISM –The genetic code (this lecture; 12a) –Synthesis of aminoacyl-tRNA (next lecture; 12b) –Translation: RNA-dependent protein synthesis...

Lecture 12a 12a/ The Genetic code Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 27: p1005-1014 PROTEIN METABOLISM –The genetic code (this lecture; 12a) –Synthesis of aminoacyl-tRNA (next lecture; 12b) –Translation: RNA-dependent protein synthesis (next lecture; 12b) – Protein processing, targeting and degradation (see lecture 13) PROTEIN SYNTHESIS IS HIGHLY ENERGY-DEMANDING, SO IT IS HIGHLY COORDINATED Protein synthesis can use up to 90% of the chemical energy of a cell Number of copies of protein produced corresponds to the number of copies needed Proteins are targeted to cellular locations Degradation keeps pace with synthesis PROTEIN SYNTHESIS IS A VERY COMPLEX PROCESS In eukaryotes, protein synthesis requires more than 300 biomolecules: – >70 ribosomal proteins – ~20 aminoacid activation enzymes – ~20 protein factors for initiation, elongation, and termination of peptides – ~100 additional enzymes for final processing – ~40 kinds of tRNAs and rRNAs THREE EARLY ADVANCES IN UNDERSTANDING PROTEIN SYNTHESIS 1) Proteins synthesized at ribosomes 2) Amino acids activated for synthesis by attachment to tRNA via aminoacyl-tRNA synthetases 3) tRNA acts as an “adapter” to translate mRNA into protein MOST RIBOSOMES ARE ATTACHED TO THE CYTOSOLIC FACE OF ENDOPLASMIC RETICULUM Electron micrograph and schematic drawing of a portion of a pancreatic cell, showing ribosomes attached to the outer (cytosolic) face of the endoplasmic reticulum (ER). The ribosomes are the numerous small dots bordering the parallel layers of membranes. ADAPTOR (tRNA) BRINGS AMINO ACID TO mRNA Since the Crick’s adaptor hypothesis, we know today that the adaptor is a tRNA: Amino acid is covalently bound at the 3’ end of the tRNA molecule and that a specific nucleotide triplet elsewhere in the tRNA interacts with a particular triplet codon in mRNA through hydrogen bonding of complementary bases. THE GENETIC CODE FOR PROTEINS CONSISTS OF TRIPLETS OF NUCLEOTIDES AND IS NON-OVERLAPPING There are 20 common, genetically encoded amino acids A four-letter code (A,T,G,C) in groups of two is insufficient (42=16) A four-letter code (A,T,G,C) in groups of three is more than sufficient (43=64) Living organisms use non-overlapping mRNA code with no punctuation OVERLAPPING VERSUS NONOVERLAPPING GENETIC CODES In a nonoverlapping code, codons (numbered consecutively) do not share nucleotides. This is the code used in mRNA. A nonoverlapping code provides high flexibility in the triplet sequence of neighboring codons and therefore in the possible amino acid sequences designated by the code. The genetic code used in all living systems is now known to be nonoverlapping. In an overlapping code, some nucleotides in the mRNA are shared by different codons. In a triplet code with maximum overlap, many nucleotides, such as the third nucleotide from the left (in red; A), are shared by three codons. Thus, the triplet sequence of the first codon limits the possible sequences for the second codon. FEATURES OF THE GENETIC CODE The code is written in the 5‘à 3’ direction The third base is less important in binding to tRNA First codon (“start” codon) establishes the open reading frame (ORF) – If reading frame is thrown off by a base or two, all subsequent codons are out of order 61 of the 64 codons code for amino acids 3 are termination codons – UAA, UGA, UAG AUG = initiation codon (start ORF) as well as a Met codon) NUCLEOTIDE CODE DICTIONARY - The codons are written in the 5′→3′ direction. - The third base of each codon (in bold type) plays a lesser role in specifying an amino acid than the first two. - The three termination codons are shaded light red, the initiation codon AUG in green. - All the amino acids except methionine and tryptophan have more than one codon (green). - In most cases, codons that specify the same amino acid differ only at the third base. DEGENERACY OF THE GENETIC CODE Most amino acids have more than one codon. Only Met and Trp have a single codon The genetic code is resistant to mutations: Some codons are less subject to causing a mutation in an amino acid sequence because of : – degeneracy of the code (there are several codons for a given amino-acid) – codon bias: certain codons for the same amino acid are used less frequently than others. THE GENETIC CODE IS UNIVERSAL, WITH A FEW EXCEPTIONS Used by prokaryotes and eukaryotes, across species Mitochondria contain DNA and use a slightly different code – UGA encodes Trp in vertebrate mitochondrial DNA (instead of STOP) – AGA/AGG encodes STOP in vertebrate mitochondrial DNA (instead of Arg) Mitochondria encode their own tRNAs, use 22 tRNAs instead of 32 used in the standard code. This is due to an unusual set of wobble rules: for example 1 tRNA recognizes 4 codon families (based on only the 2 first bases of the codon) PAIRING OF CODON in mRNA AND ANTICODON in tRNA Alignement of the two RNAs (tRNA and mRNA) is antiparallel The codon sequence is complementary with the anticodon sequence The codon in mRNA base pairs with the anticodon in mRNA via hydrogen bonding The alignment of two RNA segments is antiparallel “WOBBLE” PAIRINGS IN tRNA WITH mRNA CAN OCCUR IN THE THIRD BASE The “wobble” hypothesis = proposes the third base of most codons pairs loosely with the corresponding anticodon base: – The third base of a codon can form non-canonical base pairs with its complement (anticodon) in tRNA – permits rapid dissociation of the tRNA from its codon during protein synthesis – Some tRNAs contain Inosinate (I), which can H-bond with U,C, and A These H-bonds are weaker and were named by Crick as “wobble” base pairs Example: In yeast, CGA, CGU, and CGC all bind to tRNAArg, which has the anticodon 3’-GCI-5’(see next slide) Note: Although sequences are usually written 5’à3’, the anticodon here is written 3’à5’ to illustrate its bonding to the mRNA codons WOBBLE ALLOWS SOME tRNAs TO RECOGNIZE MORE THAN ONE CODON The anticodons in some tRNAs include the nucleotide inosinate (designated I) Inosinate can form weak hydrogen bonds with A, U, and C Pairing relationship of codon and anticodon: 3 different codons pairing possible with inosinate WOBBLE ALLOWS SOME tRNAs TO RECOGNIZE MORE THAN ONE CODON The first two bases of the codon form strong Watson-Crick base pairs with the anticodon – confers most of the coding specificity the first base of the anticodon (read in the 5′⟶3′ direction) determines the number of codons recognized by the tRNA THE RELATIONSHIPS OF THE WOBBLE HYPOTHESIS When an amino acid is specified by several different codons, the codons that differ in either of the first two bases require different tRNAs At least 32 tRNAs are required to translate all 61 codons (31 to encode the amino acids, 1 for initiation) Most cells have more tRNAs than the 32 tRNAs required to translate all codons E. coli has 47 different tRNA genes in the genome. Many are in multiple copies à 86 tRNA genes in total THE GENETIC CODE IS RESISTANT TO MUTATION Degenerate code allows certain mutations to still code for the same amino acid – “silent” mutations―different nucleotide in DNA but same amino acid in protein Mutation in first base of a codon usually produces a conservative substitution – Example: GUU à Val, but AUU àLeu Codon bias (certain codons for the same aminoacid used less frequently than others) minimizes the effect of mutations TRANSLATIONAL FRAMESHIFTING AFFECTS HOW THE CODE IS READ Translational frameshifting = “hiccupping” of ribosomes at a certain point in the translation to change the reading frame – Allows more than 2 related but distinct proteins to be produced from a single transcript – occurs during translation for the overlapping gag and pol genes of the retrovirus Rous sarcoma virus (the ribosome bypasses the UAG stop codon at the end of gag gene Polyprotein gag-pol is then processed (see RNA lecture) UUUAU is a slippery site for the ribosome so that it shifts the reading frame and bypasses the stop codon, making polyprotein gag-pol that is then processed (see RNA lecture) Determined also by secondary structure of RNA TRANSLATIONAL FRAMESHIFTING AFFECTS HOW THE CODE IS READ Slippery sequence (biased by availability of tRNA) RNA secondary structure (pauses the ribosome) A graphical representation of the HIV1 frameshift signal: A −1 frameshift in the slippery sequence region results in translation of the pol instead of the gag open reading frame (ORF). Both gag and pol proteins are required for reverse transcriptase, which is essential to HIV1 replication (see previous slides and JC on HIV) from Wikipedia SOME mRNAs ARE EDITED BEFORE PROTEIN SYNTHESIS RNA Editing involves alteration, addition, or deletion of nucleotides in mRNA in a manner that affects the meaning of the transcript during translation Editing uses guide RNAs (gRNAs) that temporarily hybridize with the mRNA and act as templates for editing Common in RNAs from mitochondrial and chloroplast genomes EXAMPLE OF RNA EDITING IN THE TRANSCRIPT OF THE CYTOCHROME OXIDASE SUBUNIT II GENE FROM TRYPANOSOMA BRUCEI MITOCHONDRIA Post-transcriptional editing inserts four U residues – revises the reading frame guide RNAs (gRNAs) = act as templates for the editing process (a) Insertion of four U residues (red) produces a revised reading frame, using of a multienzymatic complex, the editosome (endonuclease, U-transferase etc.) (b) A special class of guide RNAs, complementary to the edited product, act as templates for the editing process. Note the presence of two G=U base pairs, signified by a blue dot to indicate nonWatson-Crick pairing.