BIOL 1107 - Gene Expression

Gene expression

the process by which DNA directs protein synthesis

2 Stages of Gene expression

-transcription

-translation

Transcription

-the synthesis of RNA using information stored in DNA

-produces a messenger RNA (mRNA)

Translation

-the synthesis of a polypeptide using information in the mRNA

-ribosomes are the sites of translation

Prokaryotes vs Eukaryotes (transc + transl)

-in prokaryotes, translation can begin while transcription is not completed

-in eukaryotes, the nuclear envelope separates the two processes

Genetic code

-translates the sequence of nucleotides from the RNA into a sequence of amino acids forming a polypeptide

-flow of information in the RNA is based on a triplet code

-the RNA is read from 5’ to 3’

Triplet Code

a series of nonoverlapping, three-nucleotide words called codons

For a given gene…

-DNA is two-stranded

• one strand is the template strand: RNA transcript will have the complementary sequence of nucleotides

• the other strand is the coding strand: it has the same sequence as the RNA (except that T replaces U)

Number of codons in genetic code

-genetic code has 64 codons

• 61 codons code for the 20 amino acids, with many redundancies

  • 1 codon always codes for a given amino acid

• among the 61 coding codons, one codes for the amino acid methionine, and is also the “start’ codon

• 3 codons do not code for anything: they are the “stop” codons

Genetic code is universal…

it has been operating very early in the history of life

What catalyzes RNA synthesis during transcription

RNA polymerase

RNA polymerase

-unwinds the DNA molecule and catalyzes RNA nucleotide bonds, follows base pairing with the template strand of DNA (except that U replaces T)

-does not require any primer

-attaches to the DNA sequence called the promoter

When does transcription end?

-Transcription ends when the RNA polymerase reaches

• a sequence called the terminator in bacteria

• a poly-A sequence in eukaryotes

Trancription unit

the stretch of DNA transcribed

Stages of Transcription

-Initiation

-Elongation

-Termination

Initiation (transcription)

-Transcription factors recognize a specific sequence in the promoter (ex: TATA box in eukaryotes)

-these factors help RNA polymerase to attach. together they form the transcription initiation complex

-RNA polymerase unwinds DNA and the trascription can start

Elongation (transcription)

-RNA Polymerase adds nucleotides to the 3’ end of the growing RNA molecule (please check this)

-a gene can be transcribed simultaneously by several polymerase

Termination (transcription)

-In bacteria, the polymerase stops at the end of the terminator. The mRNA can be translated without further modification

-in eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence (ex, a long sequence with only A nucleotides). past this sequence, the RNA is released, and the polymerase detaches

RNA modification

-in eukaryotic cells, transcribed RNA (pre-mRNA ) is processed before it is dispatched to the cytoplasm

-the 5’ end receives a modified nucleotide 5’ cap

-the 3’ end gets a poly-A tail

• the cap and tail facilitate export to the cytoplasm, help ribosome to attach, and protect against hydrolytic enzymes

Introns and Exons (RNA modification)

-non-coding sequences (introns) separate coding sequences (exons)

• introns are removed through RNA splicing (with the help of spliceosomes)

  • introns can be removed in different ways, generating several mRNA from the same gene: this is alternative splicing

Translation involves…

-Transfer RNA (tRNA)

-ribosomes

Transfer RNA (tRNA)

-they can bind with a given amino acid at the 3’ end and they have an anticodon triplet that matches a given codon on the mRNA

• an enzyme (aminoacyl-tRNA synthetase) matches a given tRNA with the corresponding amino acid (that is charged and become more reactive)

Ribosomes

facilitate the coupling between tRNA anticodons and mRNA codons during protein synthesis

Ribosome structure

-they are composed of two subunits (large and small) made of proteins and ribosomal RNA (rRNA from the nucleus)

-ribosomes have three binding sites for tRNA

• P site holds the tRNA that carries the growing polypeptide chain

• A site holds the tRNA that carries the next amino acid

• E site (exit site) where discharged tRNA leave the ribosome

3 steps of translation

-Initiation

-Elongation

-Termination

Initiation (translation)

-the small ribosomal subunit binds with mRNA and a special tRNA (initiator tRNA)

-the initiator mRNA carries the amino acid methionine and recognize the start codon (AUG)

-the large ribosomal subunit attaches and completes the translation initiation complex

-the initiator tRNA is the only one that can enter directly into the P site of the ribosome

Elongation (translation)

-new tRNA enters the A site: codon recognition

-a new amino acid is added at the C-terminus of the growing polypeptide chain: peptide bond formation

-ribosome and mRNA move relatively to each other: translocation

• the tRNA in site A moves into P

• the tRNA in site P moves into E and leaves the ribosome

-the process repeats

-translation proceeds from 5’ to 3’

Termination (translation)

-when a stop codon reaches site A, there is no tRNA matching the codon

-a release factor enters the site A and causes the translation complex to come apart. the polypeptide is released

After translation…

-most polypeptides are modified (post-translational modifications) or targeted to specific organelles

• proteins are targeted to the endoplasmic reticulum if they carry a signal peptide

• a signal-recognition particle (SRP) binds to the signal peptide and guides the peptide to the endoplasmic reticulum membrane

Bacteria vs Prokaryotes (translation)

in bacteria, transcription and translation can be coupled directly, while eukaryotes separate the two processes (due to nuclear envelope)

Many ribosomes…

can translate a single mRNA simultaneously

Mutations

changes in the cell’s genetic information

Point mutation

-one nucleotide pair change in a gene sequence

• it can lead to the production of abnormal protein

Nucleotide-pair substitution

replaces one pair of nucleotides by another one

Silent mutations

have no effect (redundancy of genetic code)

Missense mutation

codes for an incorrect amino acid

Nonsense mutation

codes for a stop codon

Insertion/Deletion

addition/loss of several nucleotides usually creates a frameshift mutation and deeply alters the structure of the protein

Mutations can occur…

during errors in DNA replication or due to the action of mutagens