Transcription and Translation

• Genetic information is stored as DNA.
• The information takes the form of the specific sequences of the four different bases.
• The DNA must be able to direct the synthesis of proteins, and there must be mechanisms for controlling when and how much of each protein is made. See Figure 11.5.
• Not all DNA is used. Some cells never use DNA that other cells use. An example is that we rarely find a hairy liver.

DNA acts as a template for the synthesis of RNA, a molecule which is complementary, anti-parallel to the DNA template, differs by having a ribose instead of deoxyribose; has a base called uracil instead of thymine, and is synthesized in the 5' to 3' direction using the DNA 3' to 5' template. See Figure 11.8.

• Only one of the strands of the double-stranded DNA molecule acts as the template for the synthesis of mRNA (the m abbreviates messenger).

• In bacteria, the mRNA is produced by an enzyme, RNA polymerase. RNA polymerase is a holoenzyme composed of two alpha and two beta subunits, and a sigma factor. The sigma factor is essential to recognition of the regions where transcription should start.

• RNA polymerase:
  • Prokaryotes have two: primase and RNA polymerase.
  • Eukaryotes have three, RNA polymerase I or A, which transcribes sequences in the nucleolar organizer; RNA polymerase II (or B), which transcribes all genes except those done by the other two; and, RNA polymerase III (or C), which transcribes the 5S ribosomal RNA, small nuclear RNAs and tRNA genes.
• RNA synthesis must start at the correct spot on the DNA and must stop at the correct spot.

• A promoter is a specific sequence of DNA that is recognized as such by RNA polymerase.
  • At this sequence, RNA polymerase proteins associate with sigma factor, and RNA synthesis begins a few bases downstream.
  • A sequence, TATAAT, is called the Pribnow Box. It is located 10 base pairs up from the point where RNA synthesis begins. It is said to be at -10bp.
  • When numbering the bases of an RNA molecule, base 1 is the first transcribed base. Each base then gets a sequential number until the final base transcribed is numbered.
• The Template strand is also called the non-coding strand. Its sequence is complementary, but different from the RNA that it is a template for. The coding strand is the strand that is not used as a template; its sequence is the same as the RNA that is synthesized, except it has deoxyribose, not ribose; and, thymine, not uracil. See Figure 11.7.

• The TATAAT sequence is called a consensus sequence for promoter regions, because it is found universally in prokaryotes.
• Sequences from different sources, that are alike or close are called consensus sequences, homologous sequences; or, sequences that have homology, a degree of homology, or are said to be conserved if partially identical or highly conserved if almost identical. See Figure 11.11.

• The TATAAT sequence of promoters do differ from each other somewhat. Some versions have a higher affinity for the RNA polymerase, affecting the binding rates and the amount of RNA synthesized. Highly effective promoter regions are called hot promoters. The hottest version of TATAAT is exactly that sequence.

• Sequence differences between other regions of the promoter region are what make it possible to control the amount and time of RNA synthesis. Different sigma factors also add to the range of regulatory possibilities (heat-shock proteins).

Initiation:

• RNA Polymerase finds promoter region. Associates loosely with dsDNA, this is called a closed promoter complex. Then a tighter complex forms with ssDNA, this is called an open promoter complex. See the melting in process Figure 11.9.

Elongation:

• The open bubble of DNA moves along, held open by the RNA polymerase complex. The RNA hangs through a region of the polymerase. In prokaryotic cells, translation will be occurring already, even before the mRNA is completed.

Termination:

• The sequence of DNA that signals RNA polymerase to stop incorporating bases into the RNA molecule is called a terminator sequence. It is within, but toward the end (downstream) of the coding region of the gene.
• In prokaryotes, there are two general types of terminators, one is called rho-dependent and the other, rho-independent. See Figure 11.13.
• The RNA sequence generated from the DNA of the rho independent terminators are inverted repeats, also called palindromic sequences.
  • These RNAs are able to form hairpin loops and other secondary structures.
  • The hairpin loop forms in the RNA molecule and this interacts with the RNA polymerase as to cause it to disassociate from the DNA molecule. Thus, RNA synthesis of each RNA molecule is terminated.
• Rho dependent terminators have a "rut" binding sequence on the mRNA, which when generated on the RNA molecule, binds a rho protein that either then moves up to the RNA polymerase molecule causing it to disconnect from the DNA, or otherwise causes the RNA to disassociate from the complex; depending on which model is being discussed. See summary diagram Figure 11.10.

• The transcript has a sequence that tells the protein synthesis machinery to start adding amino acids.
  • This is called the start codon; AUG is the start codon almost universally (GUG can also code start in E. Coli).
  • The sequence of the RNA molecule, also called the transcript, upstream (5') of the start codon, is called the leader.
• The transcript has a stop codon that tells the protein synthesis machinery to stop adding the amino acids.
  • There are three possible codons for this sequence: UAA, UAG, or UGA.
  • The portion of the RNA molecule that is downstream from the stop codon (sometimes called a nonsense codon) is called the trailer sequence.

• There are three main types of RNA in prokaryotes: tRNA, rRNA, mRNA.

The general theme is the same as in prokaryotes except for a few major differences.

• In prokaryotes, translation is not physically separated from transcription. In fact, translation starts before transcription even ends. In eukaryotes, transcription occurs in the nucleus, whilst translation occurs exclusively outside the nucleus in the cytosol. Also, some translation occurs in mitochondria.

• In eukaryotes, there are even more sigma-like factors.

• There are post-transcriptional modifications that are unique to eukaryotes.
  • A poly-Adenosine tail is added post-transcription.
    • The longer the tail, the longer the transcript usually survives in the cytosol.
    • It is kind of like a fuse.
    • Exonucleases begin to digest from the 3' end of the transcript, but digesting the poly A tail doesn't affect the coding regions of the transcript.
    • The poly-A tail has been a useful reality for cloning mRNAs from eukaryotes.
  • A cap is put on the 5' end. It is called a 7' methylguanosine cap. It is thought to protect against RNA exonuclease degradation. See Figure 11.15.
  • Genes with intervening sequences called introns are commonly found.
    • The RNAs have these sequences prior to processing.
    • These pre-mRNAs, called hnRNAs, have protein coding regions called exons, as well.
    • The RNA is processed, usually by snRNPs ("snurps") (in mitochondria, spliceosomes), and the exon pieces are attached together, whilst the introns are removed.
    • The system provides another point of control for gene expression, because until the RNA is processed, it will not move into the cytosol; and, it might not get processed, or not until the correct snRNP complex is present.
    • Some have alternative splicing for the same RNA, depending on what splicing system is active within the cell. Mammalian immunoglobulin genes have such a system.
    • It is thought that exons are occasionally swapped between different genes, over time, sometimes creating a new and useful combination.
    • The proteins are therefor hybrid of previously existing ones. Still introns are weird! See Figures 11.4, 11.23, 11.26 and 11.28.

Translation is about directing protein synthesis. In its simplest form, protein synthesis would be diagrammed like this: see Figure 12.2.

• For a basic review of protein structure see Figures 12.3 and 12.4.
• For an overview of translation, see Figure 12.6.
• The mRNA is used as instructions for the synthesis of the protein. This requires two general types of mechanisms:
  • tRNA: Transfer RNA is a specialized form of RNA that brings the correct amino acid to the protein synthesis machinery and has an anticodon that aligns with the mRNA codon.
  • Synthesis requires tRNA-amino acids, a transcript, ribosomes, energy, and other factors.
  • Ribosomes: Ribosomes are the organelles where protein synthesis occurs.
    • Ribosomes are composed of 2 subunits.
    • In E. coli, a 50S and a 30S subunit makeup the protein-rRNA component of the ribosome.
    • The 30S has an associated 16S rRNA molecule.
    • The 50S has two rRNAs, a 23S and a 5S. See Figure 12.7.
    • In prokaryotes, one RNA transcript contains different rRNAs. The transcript is then cleaved into the different molecules.
    • This assures that the amount of each rRNA species is always equal. See Figure 12.10.

• Transfer RNA carries a certain amino acid to the ribosomes. There are specific tRNAs for each different amino acid. See Figure 12.11 and 12.12.

• The correct amino acid is attached to its tRNA by a specific aminoacyl-tRNA synthetase (ATS).
• Every amino acid has its own specific ATS.
• Some ATSes can recognize more than one of the different tRNAs that are for a given amino acid.

• (A codon is 3 bases that read a specific amino acid, or a start or stop.)
• The general shapes of each of the different tRNAs are extremely similar.
• The tRNAs contain unusual nucleotides; these are created by modifications that occur subsequent to transcription.
• The tRNAs are recognizable by their hairpin looping structure.
• The tRNA must be recognized by the appropriate ATS in order to get charged with the correct amino acid and must complement the correct mRNA codon(s).
• Each tRNA contains an anticodon sequence, which is complementary (or close to being complementary) to the RNA codon.
• Transfer RNA: The purpose is to match the correct amino acid to the mRNA codon for that amino acid. In bacteria there are 20 aminoacyl-tRNA synthetases, one for each amino acid. There are 50 tRNA molecules and 64 mRNA codons, so each ATS recognizes more than one tRNA as being correct and most tRNAs can base pair in the ribosome with more than one mRNA codon.

What is the nature of the genetic code?

• After it was determined that DNA was the genetic material, many felt there had to be a simple code.
  • One for one, would not work. There are not enough different codons for the number of different amino acids. Only four different codons would be possible.
• Two? Two generate 16 codons: 4² = 16, but still not enough.

• However, three is plenty: 3⁴ = 64. It was guessed that three was the magic number for the length of a codon, and this was later verified. Three is the length of the codons for all life on earth.

• Initial evidence came from studies on frame-shift mutants. If you insert one base into a gene's sequence, the function of the gene fails; insert two and function still fails; but, three and the function returns. The same was true for deletions. These data are consistent with the code being triplet.

• Does the code include punctuation and overlaps? The concern was that sometimes one base pair change altered the protein, substantially. It was found these were caused by the generation of stop codons. Mostly, one nucleotide base pair change results in a single amino acid change, which is consistent with no punctuation, and no overlap.

• What is the code, i.e., which codons code for which amino acids?
  • An artificial system was put into place.
    • It became possible to make synthetic mRNA using polynucleotide phosphorylase.
    • Second, an in vitro translation system was created. The great luck was that translation could proceed without a starting region or any other upstream recognition.
  • When only U was added, which would create the UUU codon, they got phenyalanine polymers; CCC generated proline polymers; GGG, glycine polymers.
• Final work was done using a tRNA filter capture system and the complete code as deciphered. See Table 12.2.

• The code is degenerative because certain amino acids are coded for by more than one codon. However, each codon only codes for one amino acid.

• The third base of the mRNA codon and the first base of the anticodon, is the least critical position for a match. This is called wobble. This wobble is for the alternate purine, or the alternate pyrimidine.

• In E. coli there are 50 tRNA molecules; however, all 60 plus mRNA codons are present. Therefore, some of the 50 must complementary base pair well enough.

• In every living organism, the codons in the mRNA mean the same corresponding amino acid. This is 99.9% true.

• Exceptions exist in mitochondria, where there is more wobble. There are only 24 tRNA for mitochondria. Still the end protein is the same as would be expected for the codons.

• Yeast mitochondria are even more different. For the CUX family, threonine is put in where the codon would typically read tryptophan. These mitochondria don't insert tryptophan into proteins.
• There are even more substantial differences for some protozoans.

• It is thought that the code was originally two bases instead of three; and, this is why there is wobble.
• Simpler systems had fewer amino acid-like molecules to code for.
• Also, the wobble tolerated by the triple code allows more tolerance to point mutations.
• I think there was always three bases used, possibly with more wobble and fewer amino acid choices.
• Once a code was developed it would be impossible to start over again, without starting from scratch.

• The building of a polypeptide, or the process of translation, occurs in three stages: 1. Initiation, 2. Elongation, and 3. Termination.

• All three stages require enzymes and other protein factors; initiation and elongation also require energy provided by GTP (a molecule closely related to ATP, the energy exchange molecule; both are the RNA form, i.e. ribose containing).

• Initiation brings together the mRNA, the first amino acid (f-methionine) attached to its tRNA, and the two ribosomal subunits.
  • An initiation complex is assembled when mRNA and a special initiator tRNA bind to a small ribosomal subunit. See Figure 12.15.
  • First, the small ribosomal subunit binds to a special initiator tRNA carrying methionine, the first amino acid to be added. The start codon 5'AUG3' codes for methionine. The first methionine added in prokaryotes is a modified methionine called formyl-methionine (f-met).
  • Next, mRNA aligns on the small ribosomal subunit. In prokaryotes, the mRNA has a specific ribosome-recognition sequence called a Shine-Dalgarno sequence at the 5' upstream end that base pairs with a complementary sequence on an rRNA.
• The bound initiator tRNA with the anticodon 5'CAU3', finds and base pairs with the initiation codon, AUG, on mRNA. This initiation codon, AUG, marks the place where translation will begin and is located just downstream from the ribosome-recognition sequence. Also, this first AUG sets the reading frame.

• Assembly of the initiation complex - the small ribosomal subunit, initiator tRNA and mRNA requires:
  • Protein initiation factors that are loosely bound to the small ribosomal subunit.
  • One GTP molecule that probably stabilizes the binding of initiation factors, and upon hydrolysis, drives the attachment of the large ribosomal subunit.
• In the second step, a large ribosomal subunit binds to the small one to form a functional ribosome.
  • The initiator tRNA fits into the P site on the ribosome.
  • The vacant A site is ready for the next aminoacyl-tRNA.
  • See Figure 12.16.

• Several proteins called elongation factors take part in this three-step cycle which adds amino acids one by one to the initial amino acid.

• Codon recognition: The mRNA codon in the A site of the ribosome forms hydrogen bonds with the anticodon of an entering tRNA carrying the next amino acid in the chain. See Figure 12.17.

• An elongation factor directs tRNA into the A site.

• Hydrolysis of GTP provides energy for this step.

• Peptide bond formation: An enzyme, peptidyl transferase, catalyzes the formation of a peptide bond between the polypeptide in the P site and the new amino acid in the A site.

• Part of the large ribosomal subunit, peptidyl transferase consists of ribosomal proteins and rRNA.

• The polypeptide separates from its tRNA and is transferred to the new amino acid carried by the tRNA in the A site.

• Translocation: The tRNA in the P site releases from the ribosome, and the tRNA in the A site is translocated to the P site.

• During this process, the codon and anticodon remain bonded, so the mRNA and the tRNA move as a unit, bringing the next codon to be translated into the A site.

• The mRNA is moved through the ribosome only in the 5' to 3' direction.

• GTP hydrolysis provides energy for each translocation step. See Figure 12.14.

• Each iteration of the elongation cycle takes about 60 milliseconds and is repeated until synthesis is complete and a termination codon reaches the ribosome's A site.
• The termination codon (stop codon) is the base triplet on the mRNA that signals to end translation. See Figure 12.19.
• The stop codons are UAA, UAG and UGA.
• Stop codons do not code for amino acids.
• Termination of translation proceeds as follows:
  • When a stop codon reaches the ribosome's A site, a protein release factor binds to the codon instead of an aminoacyl-tRNA.
  • The release factor causes peptidyl transferase to hydrolyze the bond between the completed polypeptide and the tRNA in the P site. This frees the polypeptide and tRNA, so they can both be release from the ribosome.
  • The two ribosomal subunits dissociate from the mRNA and separate back into a small and a large subunit. This link is for a .mov movie. It's too big to download at home but can be viewed here at school, or from your course CD.

Single ribosomes can make average-sized polypeptides in less than a minute. In prokaryotes, clusters of ribosomes simultaneously translate an mRNA.

Polyribosome: A cluster of Ribosomes simultaneously translating an mRNA molecule.

• Once a ribosome passes the initiation codon, a second ribosome can attach to the mRNA.

• Thus, several ribosomes may translate an mRNA at once, making many copies of a polypeptide.

• Different regions for association of the ribosomes exist on the transcript (the mRNA).

• Called Shine Dalgarno (SD) sequences, they are recognized by the ribosomes, where the 30S starts to associate.

• There is a SD just upstream from the start codon for each of the protein coding regions on the polycistronic transcript.

• Transcription and translation are coupled in prokaryotic cells.

• The first amino acid in all prokaryotes' proteins is fMet (AUG calls for met, but if first, then fMet).

• Then the process of elongation occurs. The charged tRNA transfers amino acids to the nascent chain.

• The stop codon then causes translation to finish. Stop does not code for an amino acid, but for a blank.

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