Protein Synthesis

• both processes require initiation and termination events
• both require elongation
  • adding nucleotides to 3' end of RNA using information in DNA as instructions
  • adding amino acids to carboxy (COOH) end of protein using information in mRNA as instructions
• only one strand of DNA is used to synthesize RNA in transcription, the other has redundant information because it's complimentary
• However, it is possible to have coding regions in both directions. This is rare and dangerous because a single mutation can knock out two genes at the same time

• mRNA contains information needed to synthesize proteins
• protein synthesis begins at start signal at near 5' end of mRNA, continues along the coding sequence towards 3' end, reaches the stop signal
• there might be extra sequence information at ends of the mRNA (called flanking sequences) that doesn't code for protein. The extra sequence at the 5' end is called a leader sequence and the extra sequence at the 3' end is called a trailer sequence.
• in prokaryotes, ribosomes can actually bind to the RNA that is still being transcribed and can begin protein synthesis; this isn't possible in eukaryotes because RNA synthesis occurs in the nucleus and proteins are synthesized in the cytoplasm
• also in prokaryotes, more than one coding region can be present on the same mRNA
  • called polycistronic mRNA
  • different coding regions will produce different proteins
  • the different coding regions will likely be related in function
  • separate initiation and termination signals are needed for each coding region
• in eukaryotes, only single coding regions on each RNA strand
• cistron- gene in prokaryotic organism that codes for specific protein

• translation machinery must recognize the correct reading frame
• cell has mechanism of reading sequence of nucleotides in groups of 3 (called codons)
• mutations can occur which result in an insertion or a deletion in the gene sequence. This can lead to frame shifts

  AUG CAA GAC ACC (normal reading)

  AUG CAG AGA CAC C (mutation whereby extra nucleotide Gis added)

ribosomes

• contain about 80 proteins and 3-5 ribosomal RNAs (rRNAs) which interact to form specific structure
• factory for protein synthesis
• ribosome reads the information along the mRNA and assembles amino acids into a protein

tRNA

• brings amino acids to the ribosome
• has site for attachment of amino acid
• has cloverleaf structure with base-paired arms, one variable arm
• has anticodon sequence to base pair with the codon along the mRNA
• different tRNAs exist for each codon
• tRNA aligns with codon and interprets (translates) information

mRNA

• source of coding information for the protein synthesis system
• contains start and stop signals for translation

initiation factors

help the ribosome, initiator tRNA, and other components assemble the at the correct location on the mRNA and ensure that protein synthesis starts in the correct reading frame

elongation factors

are responsible for moving the ribosome along the mRNA and maintain the correct reading frame. Facilitate removal of "used" tRNAs and bringing in "new" tRNAs

termination factors

termination factors recognize the stop codons and release proteins and ribosomes

energy source

ATP or GTP which are synthesized in the mitochondria

see figure 3.7 in text

• process occurs in ribosomes
• tRNA is "charged"with the amino acid it is supposed to carry by an enzyme that recognizes both the amino acid and the correct tRNA. This enzyme recognizes tRNA by length of variable arm of tRNA in addition to the tRNA sequence
• charged tRNA is brought to the ribosome with its amino acid attached and aligns with the mRNA by matching its anticodon with the next codon on the mRNA
• ribosome join adjacent amino acids together to assemble the protein chain
• the amino acid on the newly arrived tRNA is joined to the growing end of the polypeptide chain joined through a peptide bond. The enzyme that makes the joining is called peptidyl synthetase and is part of the ribosome.
• after donating its amino acid the tRNA is released from the ribosome
• the ribosome then translocates (moves) to the next codon
• a new tRNA brings in the new AA and the process is continued
• when a stop codon is reached, the process ceases and termination factors cause the release of the completed polypeptide chain and components of the protein synthesizing machinery

Mutations in elongation factors may cause ribosome to shift incorrectly (more or fewer than three nucleotides). This leads to the synthesis of wrong or useless proteins

In prokaryotes translation and transcription can occur simultaneously. This produces a direct connection between the two processes.

A pulse chase experiment allows components of the cell to be observed. First cells are placed in a medium of radioactive amino acids for about five minutes. This labels all proteins synthesized during that time period. This is the "pulse" phase of the experiment. The cells are then removed from the radioactive medium, placed into non-radioactive medium and monitored. A piece of x-ray film is placed over the cell culture at different times during the "chase" period. In parallele cultures, cells are viewed after zero, five, ten and twenty minutes. This is referred to as chasing the radioactivity. At first the radioactive material will be in the endoplasmic reticulum. It will then move to the Golgi. Next vesicles will start to be radioactive, and finally the radioactivity will be secreted by the vesicles outside the cell.