Lecture 19

Lecture 19 Textbook Notes

17.5 Ribosome Structure and Function in Translation

• translation of each mRNA codon into the next amino acid begins when anticodon of an aminoacyl tRNA binds to the codon

• ribosomes contain many proteins and rRNAs

  • small subunit — holds the mRNA

  • large subunit — where peptide bonds are formed

• three step protein synthesis

  1. aminoacyl tRNA diffuses into the A site, it its anticodon matches the mRNA codon, it stays

  2. peptide bond forms between aminoacyl held by aminoacyl tRNA in the A site and polypeptide held in P site

  3. ribosome moves relative to mRNA by one codon, all 3 tRNAS are shifttee one position. tRNA in E site exits

initiating translation

• translation begins when a section of rRNA in a small subunit binds to a complementary sequence on an mRNA

  • Shine-Dalgarno site — ribosome binding site, about 6 nucleotides upstream from start codon (AUG)

• initiation factors — help prep ribosome for translation and bind the first aminoacetyl tRNA to ribosome

• three steps in bacteria translation inititation

  1. mRNA binds to a small ribosomal subunit

  2. initiator tRNA with a f-Met binds to start codon

  3. large subunit binds

Elongation-Extending Polypeptide

• E and A sites are empty at beginning

• ribosome is a ribozyme

• translocation — process where ribosome moves one codon down

  • requires an elongation factor protein, uses the hydrolyzation of GTP as energy

• three steps in elongation:

  1. arrival of aminoacyl tRNA

  2. peptide-bond formation

  3. translocation

Terminating Translation

• when translocating ribosomes reach one of the three stop codons, the release factor recognizes it and fills the A site

• release factor triggers hydrolysis of bond that links tRNA in P site to polypeptide, freeing the polypeptide

Lecture Slides

Amino Acid Codons

• only 2 amino acids are specified by 1 codon each

• most amino acids are specified by 4 or even as many as 6 different codons

• 3 codons specify the termination of protein synthesis

• the genetic code is nearly universal

• codons are part of the nucleotide sequence of a mRNA

  • they determine the order in which amino acids are linked together to form a protein

  • but amino acids do NOT directly contact the mRNA

    • they are held onto mRNA by tNRA molecules

  • tRNAs act as “bridges” between amino acids and mRNA

    • short (75-80 nucleotide) single stranded RNA, but it folds up on itself by intrastrand base pairing

      • 2-D structure resembles a clover leaf

      • two most important parts are at opposite ends, in regions of unpaired nucleotides

• tRNA structure:

  • amino acid attachment site

  • anticodon (antiparallel, complementary)

• the binding of an amino acid to a tRNA molecule is very specific

  • requires input of energy provided by hydrolysis of ATP to AMP and pyrophosphate (PPi)

  • “charging” reaction catalyzed by enzymes called aminoacyl tRNA synthetases

    • a different aminoacyl tRNA synthetase for each amino acid

    • ex: histidyl tRNA synthetase addes His to its tRNA

    • each enzyme recognizes both “ends” of tRNA, which enables it to identify if this is a tRNA that it is “allowed” to add its amino acid to

• charged tRNA hybridized to mRNA:

  1. the 3’ end of a tRNA attaches to an amino acid

  2. tRNA contains regions of complementary base pairing (intrastrand base pairing)

  3. the anti codon pairs with a codon on the mRNA

Ribosomes: the sites of protein synthesis

• complexes of ribosomal RNA (rRNA) and ribosomal proteins

• function in the cytoplasm of all living organisms

• “platforms” for protein synthesis

• functionally identical, but structurally different between bacteria and eukaryotes

  • differences are important, because many antibiotics work by specifically inhibiting bacterial ribosomes

ribosome components

• prokaryotic ribosome — 70S

  • large subunit — 50S

    • 5S rRNA

    • 23S rRNA

    • ~34 proteins

  • small subunit — 30S

    • 16S rRNA

    • 21 proteins

• eukaryotic ribosome

  • large subunit — 60S

    • 5.8S rRNA

    • 5S rRNA

    • 28S rRNA

    • 49 proteins

  • small subunit — 40S

    • 18S rRNA

    • ~33 proteins

Initiation of Translation

• what is needed?

  • mRNA

  • small ribosomal subunit

  • first charged tRNA (always charged with Met)

  • accessory proteins called initiation factors

  • energy in the form of ATP

• AUG is called the initiator codon

  • it beings or “initiates” all protein chains

  • codes for special modified Met (N-formyl-methionine, or fMet) in bacteria, unmodified Met in eukaryotes

  • AUG can be found in other codons, but regular Met is added in bacteria (fMet only starts chain)

  • archaea have met has first amino acid

• steps in bacteria

  1. mRNA binds to small subunit of ribosome at ribosome binding site (Shine-Dalgarno site)

     • initiation factors help

  2. initiator aminoacyl tRNA binds to start codon

  3. large subunit of ribosome binds, completing ribosome complex

• requires

  • mRNA

  • small ribosomal subunit (30S in bacteria, 40S in eukaryotes)

  • first charged tRNA (fMet-tRNA^fMet in bacteria, Met-tRNA^Met in eukaryotes)

    • superscript indicates what amino acid that tRNA should carry

  • GTP and various initiation factors (number and names differ from bacteria to eukaryotes, by general function is similar)

• produces

  • a completed initiation complex (70s in bacteria, 80s in eukaryotes)

  • an occupied P (peptide) site and an open A (aminoacyl) site and E (exit) site

    • initiator tRNA occupies the P site

    • all other tRNAs enter into the A site

• how does a ribosome distinguish an initiator codon from an internal methionine codon?

  • by recognizing a specific sequence around the start codon — Shine-Dalgarno

  • recognized by the initiator tRNA

  • method of distinguishing differs between bacteria and eukaryotes

  • eukaryotes: ribosome scans from 5’ cap until it reaches the first AUG start codon

• in bacteria

  • Shine-Dalgarno sequence (aka ribosome binding site, or RBS) serves to align initiator AUG with initiator tRNA

  • bacterial mRNAs can have multiple RBS

  • bacterial mRNAs that carry information for multiple protein products are called polycistronic

• in eukaryotes

  • small ribosomal subunit (40S) binds to cap and finds nearest AUG

  • eukaryotes do not have multiple proteins per mRNA — monocistronic