Lecture 18

Textbook Notes

16.3 The Genetic Code

• the genetic code — the rules that specify the relationship between sequence of nucleotides and sequence of amino acids in protein

• triplet code — 3 base code

  • codon — three bases that specifies a particular amino acid

• reading frame — series of codons that specify a sequence of amino acids

• start codon — AUG signals that protein synthesis should begin at that point on the mRNA molecule, codes for methionine

• stop codons — UAA, UAG, UGA, do not code for any amino acid

• IMPORTANT PROPERTIES

  1. the code is redundant — all amino acids (except methionine and tryptophan) are coded for by more than one codon

  2. code is unambiguous — a given codon never codes for more than one amino acid

  3. code is non-overlapping — once the ribosome reads 1st codon, reading frame is established

  4. code is (nearly) universal — all codons specify the same amino acids in almost all organisms

  5. code is conservative — when several codons code for the same amino acid, the first two bases are usually identical

17.3 An Intro to Translation

• ribosomes — site of protein synthesis

• polyribosome — two or more ribosomes simultaneously translating one mRNA

• transcription/translation coupling only occurs in bacteria, because no nuclear envelope

17.4 The Structure/Function of tRNA

• aminoacyl tRNA — tRNA with an amino acid attached

Structure of tRNAs

• relatively short sequences, about 75-95 nucleotides

• forms stem-loop structures with itself

• CCA sequence at 3’ end of each tRNA is the site for amino acid attachment

• loop opposite of attachment site has an anticodon, antiparallel as well

How do Amino Acids Get Attached?

• ATP required to attach an amino acid to tRNA

• aminoacyl-tRNA synthetases catalyze the addition of amino acids to tRNAs

• for each unique amino acid, there is a different aminoacyl-tRNA synthetase and one or a few tRNAs

• each aminoacyl-tRNA synthetase has a binding site for a particular amino acid and a particular tRNA

• wobble pairing — nonstandard base pairing between nucleotide in the 3rd position of a codon and corresponding nucleotide in the anticodon of a tRNA

  • allows one tRNA to read more than one codon, so about 40 tRNAs and translate all 61 codons

Lecture Notes

Intron removal and exon splicing (part of eukaryotic mRNA processing)

• introns vary widely in length (tens to tens of thousands of bases)

• occurs post-transcriptionally (after RNA polymerase finishes that particular section), but usually concurrently (RNA polymerase doesn’t have to have finished the whole mRNA segment yet)

• must be completed prior to mRNA export out of the nucleus

• conserved sequences at both ends mark splice pointsl rest of intron sequence not directly part of removal but may play regulatory roles

• carried out by combinations of small nuclear RNA molecules and proteins (snRNPS), which precisely remove intron and join ends of exons

• steps

  1. U1 snRNP binds to pre-mRNA (at 5’ splice point)

  2. U2 snRNP binds to pre-mRNA closer to 3’ end

  3. U4/U6 and U5 snRNPs join

  4. RNA is cleaved at 5’ splice site and lariat is formed

  5. RNA is cleaved at 3’ splice site, and the two exons are joined

  6. excised intron is degraded to nucleotides

• inaccurate splicing leads to defective proteins

  • thalassemias (defective hemoglobin) often caused by inaccurate splicing of introns

  • mutations in DNA carried over to RNA, and intron/exon junction not recognized by snRNPs

  • intron not removed

  • “false” splice sites then used by snRNPs, removing necessary exon sequences → defective protein

Alternative splicing

• human genes assemble their coding regions in an extensive array of combinations

• just because all exons are present in the primary mRNA does not mean they all have to be used

• different mature mRNA = different protein

• between 50%-90% of all human genes are alternatively spliced

  • number of different transcripts from a given gene ranges from 2 to hundreds or thousands in humans (more in other species)

  • average in humans is 3-4 different transcripts per gene

• accounts for 100,000 proteins from 21,000 genes

• so genome size and complexity do not have correlation in eukaryotes

Additional processing of eukaryotic mRNAs

• modifying 5’ end of mRNA:

  • RNA 5’-triphosphatase removes a phosphate group

  • guanylyl transferase hydrolyzes GTP, removing two phosphate groups. GMP is attached to 5’ end, and the PPi is released

  • methyltransferase attaches a methyl group, creating a 7-methylguanosine cap

  • so there is three phosphate groups, but they are not free, thereby preventing nucleases from degrading the 5’ end of mRNA

• modifying 3’ end of mRNA:

  • poly(A) tail added to 3’ end posttranscriptionally by poly-A polymerase

  • basically the 3’ end is cleaved by ribonuclease as the RNA polymerase is done transcriptioning, poly-A polymerase adds As onto 3’ end, creating a poly(A) tail

• function of 5’ cap:

  • allows recognition of “start signal” for translation

  • provides some stability to mRNA (protection from degradation by RNases)

• function of poly-A tail

  • provides some (temporary) stability to mRNA (protect it from degradation by RNases)

  • aids in allowing multiple copies of a protein to be efficiently made from a single eukaryotic mRNA

Summary of mRNA processing in eukaryotes

• DNA template has exon-intron-exon-intron-exon…

  • coding strand is 5’ → 3’

  • template strand is 3’ → 5’

• transcription creates an initial transcript 5’ → 3’

• excision, splicing the introns out

• capping at 5’ end

• poly-A addition at 3’ end

• mature RNA: 5’-(m^7Gppp cap)—joined exons—(poly A tail)-3’

• in bacteria

  • all of this is happening in cytosol

  • DNA -(transcription)→ mRNA → ribosome -(translation)→ polypeptide

• in eukaryotes

  • in nucleus: DNA -(transcription)→ pre-mRNA -(RNA processing)→ mRNA

  • export mRNA out the nucleus into the cytosol

  • mRNA → ribosome -(translation)→ polypeptide

Translation: turning mRNA into Protein

• nucleotide sequence (ACGU) of mRNA has information for making protein

  • but proteins are made of amino acids

• 3 nucleotides per amino acid is the minimum nunber to account for the 20 amino acids we already know about

deciphering the genetic code

• related experiments uncovered what each of the 64 codons specified

  • 61 specify an amino acid

  • 3 specify the end of the protein (stop codons)

• if 61 specify amino acids, and there are only 20 amino acids, what are the 41 used for

  • genetic code is degenerate or redundant

  • most amino acids can be specified by multiple codons

  • genetic code is unambiguous — one codon can only specify one amino acid

  • genetic code is non-overlapping