Gene Expression, Transcription, and Translation

Introduction

• This chapter covers gene expression, specifically transcription and translation.
• Key topics include the machinery of transcription, mRNA processing, and the machinery of translation.

What Does DNA Do?

• DNA serves as the molecular blueprint for every cell.
• DNA contains recipes for:
  • Proteins
  • RNA
  • More DNA
• A gene is a sequence of DNA that codes for a protein.

Gene Expression

• Gene expression is the process of using a gene to make a protein or RNA product.
• Proteins and RNA are the "molecular workers" of the cell.
• Different cell types produce different proteins and RNAs at different times.
• Controlling gene expression allows cells to manage their "workforce."

The Central Dogma of Molecular Biology

• The central dogma outlines the flow of genetic information:
  • DNA → RNA → Protein
• Transcription:
  • An RNA copy of a gene is made from DNA.
• Translation:
  • The ribosome reads the RNA sequence and makes a chain of amino acids.

Analogy for DNA, RNA, and Ribosomes

• DNA = recipes for proteins
• RNA = Xerox copy of 1 recipe (1 protein)
• Ribosome = Chef (Makes proteins from the recipe)
• Ingredients = Amino acids
• Protein folds either inside or outside the cell nucleus, either in the cytoplasm or endoplasmic reticulum (ER).
• Transcription involves mRNA, tRNA, and a chef’s assistant (rRNA).

Prokaryotes vs. Eukaryotes

• Prokaryotes:
  • The chromosome is in the cytoplasm.
  • Genes for similar proteins are often located together in the genome.
  • Transcription and translation occur simultaneously within the same compartment.
• Eukaryotes:
  • DNA is in the nucleus, and ribosomes are in the cytoplasm.
  • Transcription occurs in the nucleus, while translation occurs in the cytoplasm or on the ER.
  • Genes for different proteins are spread throughout the genome.

Transcription and Translation Overview

• The process involves converting DNA to RNA (transcription) and then RNA to a polypeptide (translation).
• The genetic code is read in triplets, with each codon specifying a particular amino acid (see chart).

Machinery of Transcription: RNA

• RNA stands for ribonucleic acid.
• RNA differs structurally from DNA:
  • RNA is typically single-stranded.
  • RNA uses the sugar ribose instead of deoxyribose.
  • RNA uses the nitrogenous base uracil (U) instead of thymine (T).

Types of RNA

• Messenger RNA (mRNA):
  • Carries DNA gene information to the ribosome.
• Ribosomal RNA (rRNA):
  • Forms part of the structure of ribosomes.
• Transfer RNA (tRNA):
  • Brings amino acids to the ribosome.

RNA Polymerase II

• RNA polymerase II:
  • Pries the DNA strands apart and lays in the RNA nucleotides.
  • Does not require primers.
  • Does not require helicase to unwind the DNA.

Phases of Transcription

• Transcription occurs in three phases:
  • Initiation
  • Elongation
  • Termination

Phase 1: Initiation

• Promoter Region:
  • DNA sequence to which RNA Polymerase attaches (3' end of gene).
  • Located just upstream of the start codon.
  • In eukaryotes, includes a TATA box.

Forming the Transcription Initiation Complex

• Proteins called transcription factors, RNA Polymerase II, and the DNA promoter form a complex.
• This complex helps RNA polymerase II connect to the 3' end of the DNA template strand and open the helix.
• If any of the proteins are missing, transcription cannot start.

Phase 2: Elongation

• RNA Polymerase II tracks along the template strand of DNA (from 3' towards 5') and matches RNA nucleotides to the DNA nucleotides.
• A pairs with U, and C pairs with G.
• The mRNA strand peels away from the DNA, and the DNA seals back up.
• Multiple RNA Polymerase II enzymes can transcribe a gene simultaneously.

Phase 3: Termination

• RNA Polymerase II stops transcribing and releases the mRNA.
• The transcription machinery falls apart.
• In eukaryotes, transcription ends a little beyond the stop codon.
• In prokaryotes, transcription ends at a special terminator sequence.

mRNA Processing

• In bacteria, no further processing is necessary before translation begins.
• In eukaryotes, the mRNA transcript must be modified before translation can begin.
• Both ends and parts of the interior of the transcript are modified.

Alteration of mRNA Ends

• Each end of an mRNA molecule is modified:
  • The 5′ end receives a modified nucleotide 5′ cap (usually guanine).
  • The 3′ end gets a poly-A tail.

Why the Modifications?

• These modifications serve several functions:
  • Facilitate the export of mRNA from the nucleus.
  • Protect mRNA from hydrolytic enzymes.
  • Help ribosomes attach to the 5′ end.

RNA Splicing

• Introns are removed from the transcript.
• Exons are the useful coding pieces of DNA that are expressed.
• Introns are non-coding regions sometimes referred to as "junk DNA."

What do Introns Do?

• Introns aren't really "junk."
• They may code for amino acids not necessary for making the specific protein needed.
• They allow the cell to make multiple, similar proteins from a single gene recipe.

Splicing Machinery

• Spliceosomes:
  • Complexes of proteins that clip out the introns.
• Ribozymes:
  • RNA molecules that function as enzymes and can splice mRNA.

The Machinery of Translation

• Involves the coordinated efforts of mRNA, tRNAs, and ribosomes.
• Consists of three phases:
  • Initiation
  • Elongation
  • Termination
• These phases have the same names as in transcription, but different processes occur.

Ribosomes

• Large complexes of proteins and rRNA.
• Made of two subunits:
  • The large subunit binds to two tRNAs and has machinery that puts together amino acids into a chain (E, P, and A sites).
  • The small subunit grips the mRNA and slides it through the ribosome while matching it with the tRNA anticodons.

tRNA

• Consists of three basic parts:
  • A folded-up piece of RNA and proteins.
  • An amino acid attached to one end of the RNA.
  • An anticodon at the opposite end of the RNA.

Anticodons

• Region of the tRNA sequence.
• A small piece of RNA three nucleotides long.
• The complementary sequence that matches the codon within the mRNA for a specific amino acid.

Anticodons are Complementary to Codons

• The anticodon exactly matches a specific mRNA codon.
• Each tRNA carries the amino acid that matches its anticodon.
• The job of the tRNA is to read the codons and exactly match them with the correct amino acids.

Phase 1: Initiation (Translation)

• Ribosomes, tRNA, and mRNA build the translation initiation complex.
  1. The mRNA binds to the small ribosomal subunit.
  2. The mRNA slides through the subunit until the first AUG (start codon) is exposed in the tRNA binding site (P).

Initiation (cont.)

3.  The first tRNA carrying methionine (and anticodon UAC) binds to the mRNA start codon.
4.  The large ribosomal subunit joins the complex.

Phase 2: Elongation (Translation)

1.  A second tRNA that matches the second mRNA codon in the sequence enters the A site and binds.
2.  The amino acid at the top of the new tRNA binds to the first amino acid via a peptide bond.

Elongation (cont.)

3.  The whole complex shifts over so that the first tRNA is now in the E site and can exit.
4.  The next tRNA enters the now empty A site.

Phase 3: Termination (Translation)

1.  A stop codon in the mRNA reaches the A site of the ribosome.
2.  The A site accepts a protein called a release factor.

Termination (cont.)

3.  The release factor causes the addition of a water molecule instead of an amino acid.
4.  This reaction releases the polypeptide, and the translation assembly then comes apart.