15: Gene Expression

Front (1): [SEQ - Outcome] What is the "Central Dogma" of molecular biology, as proposed by Francis Crick?

• What is the flow of information?

• What are the two main processes called?

• Flow: DNA —> RNA —> Protein (This flow is unidirectional).

• Processes:

  1. Transcription: The synthesis of an RNA strand using a DNA template (DNA —> RNA).

  2. Translation: The synthesis of a polypeptide (protein) using the RNA sequence to direct the amino acid sequence (RNA —> Protein).

Compare and contrast DNA and RNA on three key points:

1. Sugar

2. Nitrogenous Bases

3. Strandedness (Structure)

• Sugar: DNA = Deoxyribose. RNA = Ribose.

• Bases: DNA = A, T (Thymine), C, G. RNA = A, U (Uracil), C, G.

• Strandedness: DNA = Almost always double-stranded. RNA = Almost always single-stranded.

List and define the three main kinds of RNA.

1. mRNA

2. rRNA

3. tRNA

• mRNA (messenger RNA): Carries the genetic "message" (the instructions for a protein) from the DNA in the nucleus to the ribosome.

• rRNA (ribosomal RNA): A structural and catalytic component of ribosomes (the "workbench" where proteins are made).

• tRNA (transfer RNA): "Transfers" a specific amino acid to the ribosome by matching its anticodon to the mRNA codon.

What is a codon? How many bases are in a codon, and how many possible combinations are there?

A codon is a sequence of 3 bases in an mRNA molecule that specifies one amino acid.

• Since there are 4 bases (A, U, C, G), there are $4^3$ (or $4 \times 4 \times 4$) = 64 possible codons.

The genetic code is unambiguous and redundant. What does this mean?

• Unambiguous: Each codon codes for only one specific amino acid (e.g., codon UUU only codes for phenylalanine).

• Redundant: Most amino acids are coded for by more than one codon (e.g., Leucine is coded for by 6 different codons). This is also related to the "wobble hypothesis.

What is a "reading frame"? What is a "frameshift mutation," and why is it so damaging?

• Frameshift Mutation: A mutation (an insertion or deletion of 1 or 2 bases) that shifts this grouping.

• Damage: It changes every single codon (and thus every amino acid) "downstream" from the mutation, almost always resulting in a non-functional protein.

  • Example (deleting the 'T'): HER EDD OGA TET HEB UG...

Describe Initiation of transcription.

• What is a promoter?

• What is RNA polymerase?

• What happens in this stage?

• Promoter: A specific DNA sequence before the gene that indicates the transcription start site. (It is not transcribed).

• RNA Polymerase: The enzyme that synthesizes the new RNA strand.

• Action: RNA polymerase binds to the promoter, unwinds the DNA helix, and prepares to begin transcription.

Describe Elongation of transcription.

• What direction is the DNA template "read"?

• What direction is the new RNA strand synthesized?

• What base pairs with Adenine (A) on the DNA template?

• RNA polymerase moves along the DNA, unwinding it.

• It "reads" the DNA template strand in the 3' → 5' direction.

• It synthesizes the new, complementary RNA strand in the 5'→ 3' direction.

• Uracil (U) (in the new RNA) pairs with Adenine (A) (on the DNA).

Why do eukaryotes need to modify their mRNA after transcription, while prokaryotes do not?

Spatial and Temporal Separation.

• Eukaryotes: Have a nucleus. Transcription happens inside the nucleus, and translation happens outside in the cytoplasm. The processes are separate. The mRNA needs to be processed and protected for its journey.

• Prokaryotes: Have no nucleus. Transcription and translation can happen simultaneously on the same mRNA molecule.

In eukaryotes, what is "pre-mRNA"? What are the two modifications made to its ends, and what are their three main purposes?

• pre-mRNA: The initial, unmodified RNA transcript.

• Modifications:

  1. A 5' cap is added.

  2. A 3' poly-A tail (a long chain of Adenines) is added.

• Purposes:

  1. Help export the mRNA from the nucleus.

  2. Help with ribosome recruitment (binding for translation).

  3. Protect the mRNA from degradation by enzymes.

What is RNA splicing? Define introns and exons.

• Splicing: A process that occurs in the nucleus to create mature mRNA.

• Introns: Intervening, internal regions that do not code for amino acids. They are removed ("spliced out").

• Exons: Expressed regions that do code for amino acids. They are "spliced together" (connected) to form the final, mature mRNA.

What is tRNA? What are its two most important functional regions?

Its job is to "transfer" a specific amino acid to the ribosome.

1. Anticodon: A 3-base loop on the tRNA that is complementary to an mRNA codon. This is how it "reads" the message.

2. Amino Acid Attachment Site: The 3' end where the correct amino acid is attached.

Describe the ribosome and its role.

• What is it made of?

• What are its three "sites"?

• What is a "ribozyme"?

• Made of: Protein and rRNA (ribosomal RNA). It has two subunits (large and small).

• Three Sites:

  • A site (Aminoacyl): "Arrival." Where the next tRNA enters.

  • P site (Peptidyl): "Polypeptide." Where the growing polypeptide chain is held.

  • E site (Exit): "Exit." Where the empty tRNA leaves.

• Ribozyme: An RNA molecule that acts as an enzyme. The rRNA in the ribosome is a ribozyme that catalyzes the formation of peptide bonds.

Describe translation Initiation.

• The small ribosomal subunit binds to the mRNA (at the 5' cap) and the first initiator tRNA (which carries Methionine).

• They scan along the mRNA until they find the start codon (AUG).

• The large ribosomal subunit associates, placing the first tRNA in the P site. The A site is now open, and initiation is complete.

What are the 3 steps of the Elongation cycle in translation? (Hint: APE sites)

• Codon Recognition: A new tRNA with the correct anticodon lands in the A site.

• Peptide Bond Formation: The polypeptide chain from the P-site tRNA is transferred and bonded to the amino acid on the A-site tRNA. (This is the ribozyme action).

• Translocation: The ribosome shifts one codon down the mRNA. The empty tRNA moves to the E site (and exits), and the tRNA holding the polypeptide moves to the P site. The A site is now open for the next tRNA.

how does translation Termination occur?

• The ribosome reaches a stop codon (UAA, UAG, or UGA) in the A site.

• Release factors (proteins), not a tRNA, bind to the stop codon.

• This binding causes the polypeptide chain to be released and all components (ribosome subunits, mRNA, and release factor) to dissociate.