mRNA Translation: Process and Components

Translation

• mRNA translation begins at the start codon: AUG.
• mRNA translation ends at stop codons.
• mRNA codons are read sequentially.
• tRNA molecules transport the necessary amino acids to the ribosome.
• tRNA molecules have an anticodon to base-pair with the codon.
• Amino acids are joined by peptide bonds

Process of Translation

• A tRNA carrying the first amino acid pairs with the mRNA start codon; the first tRNA moves to the P site and the tRNA carrying the second amino acid enters the A site.
• The second mRNA codon pairs with a tRNA that carries the second amino acid at the A site, and the first and second amino acids are joined by a peptide bond.
• The ribosome moves along the mRNA until the second tRNA is in the P site, then the next codon is brought into the A site and the first tRNA occupies the E site.
• A second amino acid joins to the third by another peptide bond when the first tRNA releases from the E site.
• The ribosome continues to move along the mRNA adding new amino acids to the polypeptide.

Simultaneous Transcription and Translation

• In bacteria, translation can begin before transcription is complete.
• Transcription is occurring in the cytoplasm where and mRNA being transcribed is available to ribosomes before transcription is complete.
• In eukaryotes, transcription occurs in the nucleus, and translation occurs in the cytoplasm.
• After RNA is made in eukaryotes, it has to be processed to generate mRNA.

Changes in Genetic Material

• DNA changes result in genetic variations which impact microbial function.
• Survival and reproduction of a microbe with a new genotype may be favored by natural or man-made environments.
• Natural selection is the survival of new genotypes.

Mutation

• Mutation is a permanent change in the DNA base sequence.
• DNA change may cause a change in the final product encoded by the gene.
• Mutations may be neutral, beneficial or harmful.
• Mutagens are agents that cause mutations.

Types of Mutations

• Mutations may be silent or neutral if they don't affect the activity of the product.
• Genetic code degeneracy can result in a silent mutation.
  • Changing the codon CAU to CAC still produces the same amino acid (Histidine).
• Mutations may affect a nonvital location of a resultant protein.

Mutations: Base Substitutions

• A base substitution is a point mutation; it changes one base in DNA.
• mRNA will carry the incorrect base in that position.
• An incorrect amino acid may be incorporated into the resulting protein.

Types of Mutations: Missense

• Missense mutation results from base substitution in DNA, resulting in an amino acid substitution in the synthesized protein.
• Sickle cell anemia is an example of a missense mutation where there is one single change in the hemoglobin protein.

Types of Mutations: Nonsense

• Nonsense mutation results from a base substitution that results in a nonsense (stop) codon.
• This prevents synthesis of a complete functional protein

Types of Mutations: Frameshift

• Frameshift mutation is the insertion or deletion of one or more nucleotide pairs in DNA.
• The shift in translational "reading frame" where the three-by-three grouping of nucleotides that is recognized as codons by the tRNAs during translation
• Causes change in many amino acids downstream from the original mutation site.

Genetic Transfer and Recombination

• Horizontal gene transfer is the transfer of genes between cells of the same generation.
• Horizontal transfer mechanisms involve a donor cell that gives some of its DNA to a recipient cell.
• Part of the donor DNA is incorporated into the recipient's DNA in recombinant form.
• It's not a frequent event; only 1% or less of an entire population is affected.
• Genetic transfer can occur through plasmids, transposons, transformation, conjugation, and transduction.

Plasmids

• Plasmids are self-replicating, circular DNA pieces outside of a chromosome.
• Plasmids are 1 to 5% the size of the bacterial chromosome.
• They're primarily found in bacteria.
• They may code for proteins that enhance a bacterium's virulence.
  • E. coli with a plasmid can cause infant diarrhea and traveler's diarrhea, where the plasmid encodes for toxin production and attachment to intestinal cells.
  • Without the plasmid, E. coli is harmless.
• F factor (conjugative plasmid) carries genes for sex pili and transfer of the plasmid.
• R factor (resistance factors) are plasmids which encode antibiotic resistance.
  • There are two sets of genes on R factors.
    • Resistance transfer factor (RTF) is a group of genes for replication and conjugation.
    • R-determinant is a resistance gene coding for production of enzymes that inactivate drugs.

Transposons

• Transposons are DNA segments that can move from one DNA region to another.
  • They contain insertion sequences (IS) that code for transposase that cuts and reseals DNA.
    • May inactivate genes if they insert within genes.
• Complex transposons genes encode antibiotic resistance.
• Known as "jumping genes."

Transformation in Bacteria

• Transformation is the process of genes that are transferred from one bacterium to another as "naked" DNA.
• It occurs when bacterial cells die and lyse, releasing their DNA.
• Other bacteria take up and incorporate fragments of that DNA into their chromosome by recombination.
• It occurs naturally among a few bacterial genera (Bacillus, Haemophilus, Neisseria, Staphylococcus, and Streptococcus).
• Competent cells can take and incorporate large pieces of donor DNA and can be artificially made in the laboratory.
• The first demonstration was in Griffith's experiment with smooth and rough strains of Streptococcus pneumoniae.
  • Smooth strains (with capsule) are virulent.
  • Rough strains (no capsule) are not virulent.
  • Rough strain took up DNA from heat-killed smooth strain.

Conjugation in Bacteria

• Conjugation is when plasmids are transferred from one bacterium to another through cell-to-cell contact.
• Attachment and transfer occurs via sex pili, encoded by a conjugative plasmid.
• Cells must be opposite mating types.
  • Donor bacteria has a conjugative plasmid (F factor), deemed F+ cells.
  • Recipient bacteria lacks the conjugative plasmid is F- cells. - After receiving the plasmid, they become F+.

Transduction in Bacteria

• DNA is transferred from a donor cell to a recipient via a bacteriophage (a virus that infects bacteria) to a recipient cell.

Introduction to Biotechnology

• Biotechnology is the use of microorganisms, cells, or cell components to make a product. -This includes foods, antibiotics, vitamins, enzymes, and vaccines.
  • It's inexpensive and natural.
• Recombinant DNA (rDNA) technology is the insertion, deletion, or modification of genes to produce desired products (genetic engineering).

Recombinant DNA Technology

• Recombination of DNA occurs naturally in microbes.
• Scientists developed artificial techniques for making rDNA for human uses.
• A gene from one organism can be inserted into the DNA of a bacterium or yeast. - Bacteria encoding human insulin genes produce insulin to treat diabetes. - The vaccine for hepatitis B is made by yeast carrying a gene for part of the hepatitis virus, which produces a viral coat protein.

Recombinant DNA Procedures

• DNA containing a gene of interest is cleaved (cut out).
• A gene of interest is inserted into a vector, which is a self-replicating DNA molecule used to transport foreign DNA into a cell. - An example is a plasmid.
• Results in recombinant DNA (vector).
• The vector needs to be put into a living cell where the recombinant vector DNA is taken up by the cell where it can multiply.
• Cells with a recombinant vector are grown in culture to form a clone.
  • A clone is a population of genetically identical cells arising from one cell; each carries the vector.
• The cell clone containing rDNA (recombinant DNA) may be used to harvest many copies of the gene of interest or express (transcribe and translate) the gene of interest to produce the protein product.
  • An example is a gene for human growth hormone (hGH) that has been inserted into E. coli bacterium.
    • Recombinant E. coli can now produce large amounts of hGH quickly and cost-effectively.

Tools of Biotechnology: Restriction Enzymes

• Restriction enzymes are enzymes in many bacteria that cut very specific DNA sequences.
• They destroy bacteriophage's DNA in bacterial cells.
• Methylated cytosines in bacteria protect their own DNA from digestion.
• DNA to be inserted and DNA of vector are cut with the same restriction enzyme, then joined using DNA ligase.

Polymerase Chain Reaction

• The polymerase chain reaction(PCR) is a method used to amplify a specific DNA segment. -It is increasing (amplifying) small quantities of DNA to for analysis.
  • PCR can make billions of copies of a target DNA within a few hours.
  • it is used for diagnostic tests for genetic diseases and detecting pathogens To carry out PCR:
  • Add primers, nucleotides, and DNA polymerase.
  • Incubate at 94°C to separate the strands.
  • Incubate at 60°C to allow primers to attach to single-stranded DNA.
  • Incubate at 72°C for DNA polymerase to copy the target DNA.
  • Repeat the heating and cooling cycle to make more copies.

Selecting a Clone

• This refers to selecting the cells carrying the gene of interest.
• Can be selected with the process of Blue-white screening
  • Uses a Plasmid vector containing ampicillin resistance gene (ampR) and p-galactosidase gene (lacZ).
  • Bacteria is then frown in media containing ampicillin and X-gal, a substrate for β-galactosidase
    • Only those cells that received the plasmid will grow.
    • Bacteria that have the recombinant plasmid (new gene inserted within the lacZ gene) will Not hydrolyze X-gal and colonies will be white.
    • Bacteria with original plasmid with intact lacZ gene (no inserted DNA) will form blue colonies.

Making a Gene Product in Bacteria

• Commonly uses E. coli.
  • Advantages in it's easily grown and known genomics are.
  • Produces endotoxins and does not secrete it's proteins.
    • Therefore, harvesting production can be ineffecient.

Making a Gene Product

• Saccharomyces cerevisiae (yeast) -Easily grown, has a larger genome than bacteria, expresses eukaryotic genes easily and continuously secret them if wanted.
• Plants allow express eukaryotic genes, are easily grown, are large-scale, and low cost.
• Mammalin cells express eukaryotic genes easily, can make products for medical use, but can be harder to grow.

Therapeutic Applications: Gene Silencing

• Gene silencing uses small interfering RNAs (siRNAs) which bind to mRNA, which is later destroyed by RNA-induced silencing complex (RISC).
• RNA interference (RNAi) inserts DNA encoding siRNA into a plasmid and transferred into a cell therefore hereditray diesease that produce abnormal proteins are treated with RNAi

Therapeutic Applications: Gene Editing

• CRISPR stands for Clustered regularly interspaced short palindromic repeats.
• CRISPR associated enzymes are found in bacteria and archaea and is in charge of destroting foreign DNA
• Uses small RNA complementary to the desired target is attached to CAS enzyme (CRISPR-associated enzyme) and DNA to be inserted.
• The Guide RNA-CAS is inserted to target cell where Inside cell then the guide RNA binds complementary DNA and the CAS then cuts target the DNA inactivating the gene. -Insertation of DNA can then be done after