BMB 462 Lecture 33 Outline of Last Lecture I. tRNA introns – the fourth intron typeII. 3’ PolyadenylationIII. Alternative polyadenylation and splicingIV. rRNA processing in bacteriaV. rRNA processing in eukaryotesVI. snoRNPsVII. Examples of Base ModificationVIII. tRNA processingIX. Processing of small RNAsX. RNA DegradationOutline of Current Lecture I. Overview of TranslationII. Determining the Genetic CodeIII. Specifics of the tRNA codeIV. tRNA pairing with mRNAV. Charging tRNAs with amino acidsCurrent LectureConcepts to remembers from previous courses/lectures:- All tRNAs end with a CCA sequence, so they all have an adenine on the 3' end. This is important for the function of Class I and Class II aminoacyl-tRNA synthetases.I. Overview of Translationa. Translation is a very important process in the cell; it requires about 200 different proteins, 3-4 rRNAs, and 40-50 tRNAs. The rRNAs and tRNAs are utilized in protein synthesis.i. Translation accounts for as much as 90% of the cell's energy use.1. Making proteins is one of the most important processes to keep the cell functioning, besides maintaining the genetic information in DNA.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.b. The goal of ribosomal activity is to create a peptide bond.i. The amino acids come to the ribosome attached to a tRNA by an ester linkage.ii. The new peptide bond is formed by the N-terminus of the incoming amino acid attacking the ester linkage to form the new peptide bond.c. The recognition of which amino acid ought to be added next is performed by the anticodon in the tRNA, which binds to the complementary codon in the mRNA.d. There are 2 sites in the ribosome where the tRNAs can bind; the peptidyl (P) site and the aminoacyl (A) site.e. The protein is synthesized from N-terminus to C-terminus as the ribosome moves5' to 3' along the mRNA.i. N is to C in the peptide chain as 5' is to 3' in a nucleotide sequence.II. Determining the Genetic Codea. A series of genetic and biochemical experiments led to the discovery of the triplet codon.i. Francis Crick conducted experiments where he was able to isolate different mutants of the bacteriophage T4. In these mutants, he found that there could either be insertions or deletions of bases that led to the mutant phenotype. These changes in sequence altered the reading frame of the codon.1. If an insertion and a deletion occurred in the sequence, 2 codons were wrong, but after that the reading frame was restored and the wild-type phenotype was expressed once more.a. Restoring the reading frame allowed all following amino acids to be added in the correct order as specified by the wild type.i. The severity of a mutation is determined by if the reading frame of the codon is preserved.b. Biochemical experimentation was used to synthesize RNA; polynucleotide phosphorylase, which is normally used to degrade mRNA, is also capable of reversing the reaction and synthesizing small segments of RNA.i. In these experiments, the scientists synthesized short RNAs in vitro with specified ratios of bases.1. i.e. in one experiment, an RNA segment with only 'A's and 'C's wassynthesized; the bases were in a 5:1 ratio of A to C.a. Because the ratio of A:C was 5:1, the scientists knew the probability of a base being an A was 5/6, or 0.83, and the probability of it being a C was 1/6, or 0.17. This was used to calculate the probability of various codons, with the codon AAA arbitrarily chosen to be assigned an expectedvalue of 100. This value could then be compared to the probabilities of other codons to determine their expected value.i. This was done by normalizing the probability of the codon under consideration to the probability of thecodon with an expected frequency of 100. (The probability for the new codon was divided by the probability for the '100' codon).ii. By comparing the expected frequencies to the observed frequencies in their experiments, the scientists were able to assign codons to individual amino acids. They couldn't tell the exact order of the bases in the triplets,but they did know how many of each base (A or C) was present.1. With a 5:1 ratio of ‘A’s to ‘C’s so the probability for the base being an A is 5/6, and the probability for C is 1/6. Thus the expected frequency of A2C could be calculated (the Expected frequency for AAA is set to 100). The expected frequency of A2C is 20. Thus they could predict the bases in the codon that were coding for asparagine.c. The next step in biochemical experiments came when scientists, such as Gobind Khorana, were able to synthesize RNA strands with defined sequences.i. Prior to this, scientists could add the different bases but couldn't determine the actual sequence; Khorana figured out a way of making RNA with a known sequence. He then determined which amino acids were added.d. Another experiment used loaded-tRNAs - tRNA molecules with a specific amino acid attached. They then added those tRNAs to ribosomes and cell extract.i. From this, they could determine which tRNA was able to bind to a trinucleotide mRNA in the ribosome. 1. Adding a defined trinucleotide, they could determine which tRNA would bind to it by separating unbound tRNA from those bound tothe ribosome.III. Specifics of the tRNA codea. All of these experiments allowed the genetic code to be solved by 1966.i. It was determined that the code was degenerate; there are more codons than amino acids.1. For the 20 amino acids, there are 61 amino-acid specifying codonsand 3 stop codons. This implied that more than one codon specified an amino acid.a. The three stop codons are UAA, UAG, and UGA. There is also one start codon, AUG, which also codes for the aminoacid methionine; every polypeptide that is made starts with a methionine. Often that methionine is cleaved off.2. Most amino acids are coded for by 2 or 4 codons, though some amino acids are an exception to that rule.a. There are irregularities though; Leu, Ser, and Arg have 6 codons, Ile has 3. Met and Trp only have one codon.IV. tRNA pairing with mRNAa. tRNAs have an anticodon that pairs with the codon in mRNA; that is how the recognition is made.i. The codon runs 5' to 3', with bases labeled -2-3, and the anticodon runs 3'to 5', pairing to the codon bases in order 3-2-1.ii. The anticodon is antiparallel and complimentary to the codon in the mRNA (much like the double stranded DNA). b. Bases 1 and
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