From Gene to Protein - Chapter 10

What we know already• We know DNA is responsible for phenotype.

• But how is DNA responsible? It codes for proteins responsible for phenotype.

• Example: Pea plants can be tall or short. Short pea plants don’t make gibberellin which is a plant growth hormone. They lack the enzyme for gibberellin synthesis. The recipe for that enzyme is lacking in their DNA

Connection between Genes and Proteins• 1909 - Garrod first suggested this connection with what he called “inborn errors in metabolism.”

• Alkaptonuria – urine contains dark chemical alkapton because body lacks enzyme to break down alkapton. ( page 188)

• He theorized that this lack of enzymes was inherited and termed it an “Inborn error of metabolism”

• Apply the Concept page 189

One gene – one polypeptide• First hypothesis: One gene gives rise to one enzyme.

• However, since not all proteins are enzymes, more accurately: one gene gives rise to one protein.

• However, since some proteins are made of more than one polypeptide chain the most current and accurate way to say this is one gene gives rise to one polypeptide.

• It’s termed the one gene-one polypeptide hypothesis and is currently being shot down because of exceptions. It’s a good general rule though.

How DNA becomes a proteinDNA is transcribed and translated to make a protein. The general steps involved are collectively called the “Central Dogma of Molecular Biology”

DNA vs RNA

4 kinds of RNA• Messenger RNA (mRNA) – DNA is copied into mRNA during transcription.

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• Transfer RNA (tRNA) used during translation to carry the correct amino acid.

• Ribosomal RNA (rRNA) – folded strands of RNA used to make a ribosome. Site of protein synthesis.

• Small Nuclear RNA(snRNA)- Combined with proteins to make a structure called a spliceosome which helps with editing the RNA molecule.

Protein Synthesis varies between prokaryotes and eukaryotes• Prokaryotes have no nucleus so the 2 steps, transcription and translation, both occur in the cytoplasm

• Eukaryotes have 3 steps, transcription and RNA processing occurs in the nucleus, and translation occurs in the cytoplasm

From DNA to Protein• Remember that our ultimate goal is a protein. We’ll cover the three steps (transcription, processing and

translation) in detail later.

• But, for now, how does mRNA code for proteins?

– There are 20 amino acids. How do you code for 20 amino acids with only 4 nucleotide bases?

– In order to get at least 20 different combinations, we have to use at least 3 nucleotides.

– Codons are blocks of 3 mRNA nucleotides that code for an amino acid.

Breaking the Genetic code• scientists have determined which codons code for which amino acids.

Nirenberg and Matthei determined that the 1st codon – amino acid match: UUU codes for phenylalanine

• They did this by taking a chain of uracil and adding it to a test tube of ribosomes and nucleotides…they got a chain of phenylalanine

• The genetic code

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Chains of mRNA come about during transcription as a copy of the DNA is made.

DNA = TTAGACTAG mRNA = AAUCUGAUC• In that chain of mRNA there are 3 codons: AAU, CUG and AUC

• Each codon can be matched up to an amino acid.

The genetic code• Same for all life…called universal for that reason.

• Code is one of the strongest supporting arguments for a common origin for all life

• Code is redundant but not ambiguous.

• A reading frame is a set of codons read correctly. The reading frame can be shifted and the results are almost always devastating.

Practice• AAU CUG CCC codes for which amino acids?

• AUG UUU UUA codes for which amino acids?

Transcription

http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/transcription.swf

RNA processing

• Before leaving the nucleus, the newly formed mRNA strand is modified by:

• Adding a 5’ cap

• Adding a poly A tail on the 3’ end

• Cutting out the introns

• Splicing exons together

• Alteration of mRNA ends

• A modified guanine is added to the 5’ end. It will help protect the molecule from degradation and it will signal the attachment to a ribosome in the cytoplasm

• A series of 50 to 200 adenines are added to the 3’ end. This is called a poly A tail. Helps mRNA to leave the nucleus, prevents degradation and signal attachment of a ribosome

Exons and Introns• Sections of DNA that code for a protein will include interspersed sections that interrupt. In mRNA they are

removed. These are called “intervening sequences” or introns. (Also called “junk DNA”)

• When introns are cut out the remaining mRNA is eventually expressed and are called exons.

mRNA splicing• Introns are cut out and exons are pasted together.

• snRNA forms a complex with other proteins to make a spliceosome which recognizes introns and cuts them out.

Translation: a closer look • The key RNA in translation is transfer RNA.

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tRNA is a chain of about 80 or so bases. Three of bases are called the anticodon.

The anticodons are complementary to the codon in mRNA.

The top of the structure has an amino acid.

• How the amino acid joins

• The amino acids (remember that there’s 20 of them) are in abundance in the cytoplasm.

• They are attached to their specific tRNA using an enzyme called aminoacyl-tRNA synthetase.

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RibosomeMade up two subunits – large and small.

It has 4 bonding sites:

1 mRNA site on the small subunit

3 for tRNA site on the large subunit. Those three are called the E, P and A sites

Three parts of translation• 1. Initiation – Energy is required. Result of initiation: translation initiation complex: mRNA strand (always starts

with AUG), the initiator tRNA (carrying methionine) in the P site and small and large unit of the ribosome

• 2. Elongation – Amino Acids are added on at a time.

a. Codon recognition: Incoming tRNA binds to the A site.

b. Peptide bond formation - A peptide bond forms between the new amino acid and the end of a growing polypeptide chain.

c. Translocation – the tRNA in the A site moves to the P site and the tRNA in the P site moves to E site and is released

• 3. Termination – elongation continues until stop codon is reached (UAA, UGA or UAG). Stop codons have a release factor that bonds to them rather than tRNA. This factor causes the chain to hydrolyse itself from the ribosomes.

Polyribosomes

Mutations• Mutations are changes in the genetic material of a cell or virus

• Point mutations involve chemical changes in just one base pair

• If the mutation occurs in a gamete or a cell that gives rise to a gamete, it can affect the entire offspring and be transmitted to future generations. These are called genetic disorders

Types of Point Mutation

Substitution– A. Silent – makes no difference in the resulting a.a. strand

– B. missense – changes one a.a. in the resulting a.a. strand.

– C. Nonsense – inserts a stop codon into the sequence effectively ending the a.a. sequence

Insertions and Deletions• Insertion – addition of one or more nucleotides

• Deletion – deletion of one or more nucleotides

• Insertion and deletion mutations are almost always devastating because it will cause a frame-shift to occur.

– Frameshift causing extensive missense.

– Frame shift causing immediate nonsense

– Insertion and deletion of 3 nucleotides, no frame shift; extra or missing amino acid.