Translation: the Synthesis of Proteins

• View animation of translation process (requires shockwave plugin)

role of mRNA

• carries codons (3-nucleotide sequences) arranged in linear fashion that code for amino acids
• also carries signals needed to tell how to recognize ribosomes, start and stop signals for decoding protein
• leader sequence on small ribosome subunit binds to complementary sequence on mRNA, allows initial formation of RNA-ribosome complex.
• mRNA is derived from initial transcript by process of RNA processing. A 5' cap consisting of a modified guanine nucleotide is added to the 5' end. A poly-A tail made of 30 to 200 adenine nucleotides is added to the 3' end.
• View first stages of RNA processing (Campbell website activity)
• RNA processing continues with the removal of portions of the coding segment that do not actually code for protein.
1. Exons: (short for "expressed") -- regions of DNA that code for amino acids.
2. Introns: (short for "intervening" or "interrupting") -- regions of DNA inside a gene, located in between exon regions, but not coding amino acids
• When RNA is transcribed from a gene, it initally contains both introns and exons, and cannot be called "messenger RNA" yet because the message is interrupted. Introns must be removed by "cut-and-paste", called RNA splicing.
• View animation of RNA splicing in eukaryote (protected).
• snRNPs ("snurps") = small ribonucleoprotein particles, found in nucleus. Composed of RNA and a few proteins. snRNPs associate to form a Spliceosome, which locates the junction of intron and exon, specifically cuts at this junction, and joins the cut ends of exons to form messenger RNA.
• View animation of spliceosome formation (Campbell website activity)
• Ribozymes: the enzymatic activity of spliceosomes was initially thought to be in the protein. However, now known to be on RNA; first example of catalytic RNA (called ribozyme for as opposed to enzyme, which is protein).
• Note: almost all genes in eukaryotes contain intron/exon organization. In some cases, amount of intron can be much larger than amount of exon DNA.
• Evolutionary importance of introns: since many proteins consist of several domains with different functions.

role of ribosomes

• acts as a "decoding box" or "tape player" for the information in mRNA
• two parts: a small subunit and a large subunit. These are separated except when attached to m-RNA
• ribosomes contain a set of ribosomal proteins and several types of ribosomal RNA (r-RNA)
• ribosomes catalyze the formation of peptide bonds; intially thought to be due to protein activity (enzyme), but now known to be due to RNA catalytic activity (ribozyme)
• View electron micrograph of ribosomes
• View ribosomes

role of tRNA

• structure: 4 loops, anticodon, AA binding site
• View model of tRNA
• ~ 60 types in bacteria (>100 in mammals)
• only 73-93 nucleotides long
• some bases modified after transcription; form "funny bases" like pseudouridine.
• extensive hairpin loops
• anticodon site: recognizes codon on mRNA
• View interactive structure of t-RNA (requires CHIME plugin)
• requires special enzyme: Amino Acyl-tRNA synthase (= activating enzyme). ATP also required
• View model of aminoacyl tRNA synthase enzyme + tRNA
• For each tRNA, there is a unique enzyme; roughly 60 different enzymes.

Initiation of Translation

• small ribosomal subunit initiates binding to mRNA
• locates 5' end of mRNA
• small subunit ribosome finds first AUG codon = start codon
• large ribosome binds
• tRNA carries the amino acid methione to first position
• View animation of translation initiation (Campbell website activity).

Elongation of Translation

• 2 adjacent sites on ribosome: P (Peptide) and A (Amino Acid) site
• A site accepts a new tRNA-AA
• P site holds existing chain
• peptide transferred from P site tRNA to A-site AA
• enzyme activity is in ribosomal RNA (ribozyme)
• also required: Energy (GTP) and elongation factors
• View animation of translation elongation (Campbell website activity)

Termination of Translation

• reach a "stop codon" UAG, UAA, or UGA. Instead of a t-RNA, a specific release factor binds.
• Complex falls apart separate polypeptide, small ribosome subunit, large ribosome subunit.
• View animation of translation termination (Campbell website activity)
• Net cost of translation: 4 phosphate bonds/amino acid added!
• Practice translating a message (Campbell website activity).

The Genetic Code

• View Genetic Code table
• 64 possible codons (3-letter nucleotide sequences): AAA, AAC, etc.
• 60 codons serve as straightforward signals for amino acids
• Some amino acids coded by a single codon
• Some amino acids coded by as many as six different codons (redundancy)
• One codon serves as universal "start": AUG (memnonic: "A, you go!")
• Three codons serve as "stop" signals: UAG, UAA, UGA (memnonic: "You are gone, You all away, You go away")

Universality

• originally thought all organisms use identical codons
• But mitochondria of eukaryotes (except plants) use slightly different assignments for a few codons. Examples:
  1. UGA = stop (univ), or amino acid Tryptophan (mitochondria from yeast, protozoans, mammals)
  2. AUA = Isoleucine (univ), or Methinonine (mitochondria from yeast, protozoans, mammals)

Gene organization in Eukaryotes

The coding potential of human DNA

• human DNA contains 6 x 10⁹ base pairs/cell = 6,000,000 kb pairs
• compare to 4700 kb pairs/E. coli, a very sophisticated bacterium. Human DNA is more than 1000x bigger!
• If all human DNA coded for proteins, would have enough for roughly 5 million different proteins
• But currently only know ~ 3000 human proteins, and estimates as to how many we truly have range from 10,000 to 100,000
• In fact, less than 5% of human DNA codes for protein!
• What does the rest of the DNA do?

Extra Functions of human DNA

• Multigene Families: some genes are represented by more than one copy, typically for products needed in large quantity by cell.
  1. Example 1: ribosomal genes (for ribosomal RNA). Copies of the same gene are clustered together in enormous number (hundreds of thousands of identical gene copies).
  2. Example 2: histone genes (for proteins that bind to DNA to make chromatin). Family of histone proteins is represented many times.
• Pseudogenes: examples of multigene families where some copies of the gene have mutated to the point where they no longer function at all in the cell.
  1. Example: globin gene family. In humans, find several slightly different globin genes that produce the hemoglobin molecules needed by fetus, embryo, and adult. But also find a cluster of genes nearly identical in base sequence, but never expressed in the life of a human.
  2. Explanation: at some time in evolutionary past, globin genes were duplicated (by gene transposition). One cluster retained the job of making functional hemoglobin. The other cluster mutated so that promoter site no longer could be recognized by RNA polymerase. Result = this gene cluster now serves no purpose, cannot make any RNA or protein, but provides evidence of an evolutionary past. Called a pseudogene because it looks like a gene, but doesn't function.
• Repetitive sequence DNA. Some regions of DNA contain short sequences repeated many thousands of times = "tandem repeats". No coding function at all.
  1. Example 1: "satellite" DNA. Sequence such as ACAAACT repeated again and again (producing ...ACAAACTACAAACTACAAACTACAAACTACAAACTACAAACT...). These regions appear to be located where the centromere forms, so this sequence must have mechanical properties that allow recognition by kinetochore and mitotic spindle.
  2. Example 2: "telomeric" DNA. Sequences such as TTAGGG repeated over and over, 250-1500 times. Found at the ends of linear chromosomes (telomeres) where RNA primase (needed to prime the synthesis of new DNA) cannot work on lagging strand. Telomeric DNA acts like a "cap" on the end of the chromosome.
     
     Why is this needed? Because of the way DNA replication works, one strand at each end of linear DNA in each chromosome cannot be completely replicated, loses about 100 nucleotides with every replication. Gradually DNA gets shorter. These telomers provide buffering so that only telomere DNA is lost. Eventually, after 60-100 rounds of replication, telomeres have been totally lost -- at that point, further DNA synthesis leads to loss of genes. Result: cells die -- cannot continue growing longer than 100 generations.
     
     How do germ cells overcome this problem? Answer: they have special enzyme, telomerase, that synthesizes new telomere DNA before every replication, continues to restore telomeres. Same enzyme is expressed in many cancer cells!

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