Protein Synthesis

Edited Notes by Leigh Eisenman, Megan Hjermstad


Transcription

Nucleotides are added to the 3' end of RNA using information in DNA as instructions. Only one strand of DNA is used to synthesize RNA in transcription, the other has redundant information because it is complimentary. In the vast majority of cases there is no overlap between the two strands. 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.

Translation

mRNA contains information needed to synthesize proteins. Protein synthesis begins at a start signal at the 5' end of the mRNA and continues along the coding sequence towards 3' end until it reaches a stop signal. There might be extra information, or untranslated regions at the ends of the mRNA (called flanking sequences) that don'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 eukaryotes, the ends contain control elements which contain signals (for example, to degrade or stabalize) which regulate the amount of translation. The mRNA might be thousands of nucleotides long.

In eukaryones there are only single coding regions on each RNA strand, but in prokaryotes more than one coding region can be present on the same mRNA. This is called polycistronic mRNA because on a single mRNA there are multiple coding sequences which will produce different proteins. The different coding regions will likely be related in function. Separate initiation and termination signals are needed to translate each coding region. A cistron is the gene in a prokaryotic organism that codes for a specific protein.

The main objective of translation is to convert the genetic information in the mRNA into a functional protein. The terms: proteins, peptides and polypeptides are used interchangeably and may be confusing. A protein is made up of one or more polypeptide chain.

There are many components functioning in translation. Ribosomes contain about 80 proteins and 3-5 ribosomal RNAs (rRNAs) which interact to form a specific structure. The ribosomes are the "factory" for protein synthesis. They are responsibile for initiation at the correct site, accurate elongation, and termination of protein synthesis. The ribosome reads information along the mRNA, coordinates bringing the amino acids together and guides assembly into proteins.

tRNA finds amino acids and carries them to the ribosome.
Yeast Phenylalanine-tRNA
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tRNAs have the same general cloverleaf shape with base-paired arms, but with one variable side arm and a different anti-codon sequence. tRNA has the unique ability to recognize a specific codon (3 letter chunk). Different tRNAs exist for each codon. The tRNA has a site for the attachment of an amino acid. At the bottom of the tRNA there is an anti-codon that can base pair with a codon on the mRNA. The tRNA aligns with the codon and brings the appropriate amino acid to the translation machinery. The amino acid it carries can be used to add to a growing polypeptide chain. For example: the codon used to add alanine can only base pair with the alanine tRNA. Otherwise the wrong amino acid would be incorporated, such as lysine. 1/100-1,000 times a mistake is made. The genetic information in the anti-codon gets priority, so the if the tRNA carries the wrong amino acid, that incorrect amino acid will be added to the growing polypeptide chain because the tRNA is recognized during transcription only by its anti-codon, not the amino acid it carries. Certain (perscription) drugs change the rate of error and make a faulty protein (purposely).

mRNA is the source of coding information for protein synthesis and contains start and stop signals for translation. Initiation factors help the ribosome, initiator tRNA, and other components assemble 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 for maintaining the correct reading frame. In addition, they facilitate the removal of "used" tRNAs and bringing in of "new" tRNAs. Termination factors recognize the stop codons and release proteins and ribosomes. The energy source for these processes is ATPor GTPwhich are synthesized in the mitochondria.

The correct reading frame is needed for translation to produce the correct protein. The cell has a mechanism for reading a sequence of nucleotides in groups of 3 (called codons). 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. For example compare to a sequence of letters in the english alphabet:

sorexthebigreddogranandsatfortheboy 

This sequence can be read as:

s ore xth ebi gre ddo gra nan dsa tfo rth ebo y
so rex the big red dog ran and sat for the boy
sor ext heb igr edd ogr ana nds atf ort heb oy

Only one of these is in the correct reading frame and therefore makes sense. The other two are nonsense. The same situation holds for reading a mRNA in the cell.

99.9% of initiation starts at an AUG codon. A special initiator tRNAmet carrying the amino acid methionine interacts with a ribosome that is in a special state at an AUG codon (no tRNA is previously bound to ribosome). A different tRNAmet is responsible for interacting with the ribosome during elongation.

Figure 3.7; p68 illustrates translation. There are two tRNA binding sites on ribosomes. The process occurs on ribosomes. tRNA is "charged" with an 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 the variable arm in addition to the tRNA sequence. The 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.

The ribosome joins 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 through a peptide bond. The enzyme that does 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 amino acid 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 the other protein synthesizing components.

Figure 3.8; p69 is a polysome (polyribosome) drawing. In this case, multiple ribosomes are attached to the same mRNA, each making a new protein. Many identical peptides can be made from the same mRNA in a short time.

In prokaryotes, translation and transcription can occur simultaneously. Ribosomes can actually bind to the RNA that is still being transcribed and simultaneously can begin protein synthesis. This shows there is a direct connection between the two processes. (This isn't possible in eukaryotes because RNA synthesis occurs in the nucleus and proteins are synthesized in the cytoplasm).

RNAase is an enzyme which digests RNA, breaking it down into nucleotides. Constant translation actually protects the RNA in bacteria from RNAase. Ribosomes protect RNA by fitting around it like a sleeve fits around an arm. mRNAs which are more actively translated in the cell are more stable because they have more ribosomes bound to them, protecting them from RNAases. A typical prokaryotic cell has a period of 20 minutes between divisions. The cell has to change in response to its environment. For example, a bacterial cell living in the presence of lactose has to change to respond to a different energy source when the lactose is gone. So the old mRNAs need to be degraded, and replaced with new mRNAs.

In polycistronic translation there are multiple cycles of start and stop for each mRNA and coordinately regulated genes. However, when some RNA viruses infect procaryotic cells, some RNA genes are not translated immediately. This is because the AUGstart codon of one of the genes is basepaired to another region on the RNA - and is therefore unavailable to the ribosomes. The only way this start codon can be used is when the base pairing is disrupted, usually as the result of translating the complementary region of the RNA (the regions that is base paired to the AUGin question). It is the movement of the ribosomes along the mRNA that disprupts regions of intrastrand base pairing

Eukaryotes often need to synthesize proteins that are secreted from the cell. The beginning of the growing protein chain binds to the endoplasmic reticululum to start the process of secretion.The newlys synthesized protein is released into the lumen of the endoplasmic reticulum, where it is modified by enzymes. The protein eventually travels through the Golgi complex where it is modified further. The protein is then packaged in vesicles from the Golgi and transported to the cell surface, where the vesicles fuse with the cell surfact to release the protein contents.

This pathway was elucidated by a pulse-chase experiment. This experiment allowed the proteins to be tracked through the cell to the outside of the cell. 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 a non-radioactive medium and monitored. The slide is coated with photographic emulsion at different times during the "chase" period. In parallel 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.


Biology 4: Genes and Society