GB1- CHapter 17

Gene Expression

Archibald Garrod

• proposed that genes control enzymes to do specific chemical reactions, ultimately affecting phenotype

• thought that inherited diseases came from an inability to synthesize a certain enzyme in the metabolic pathway

ex) Alkaptonuria- black urine due to inability to break down tyrosine and phenylalanine

George Beadle and Edward Tatum

• made Neurospora Crassa (bread mold) mutants by exposing them to X Ray so that it can’t survive off just the minimal medium. The mutants were placed in minimum mediums+ one nutrient, and whichever nutrient presence caused it to grow, was missing from the mutant.

Adrian Srb and Norman Horowitz

• identified three arginine deficient mutants that each lacked a different enzyme to synthesize arginine

• “One gene One enzyme hypothesis”

One gene one enzyme hypothesis

hypothesis that a particular gene directs a particular enzyme

• not all proteins are enzymes, so it was renamed to “One gene, one protein hypothesis”. proteins are made up of multiple polypeptides, so it was then renamed to “One gene, one polypeptide” hypothesis.

Transcription

the synthesis of mRNA using information in DNA

Translation

polypeptide synthesis using mRNA information

• site of translation: ribosomes

central dogma

flow of information from DNA to RNA to protein

# of amino acids, # of nucleotide bases

20 amino acids, 4 nucleotide bases

codon

nonoverlapping, three nucleotide letters on mRNA that translate to amino acids

start codon- AUG

64 codons- 61 for amino acid coding

3 stop codons UAA UAG UGA

• redundant but not ambiguous. Multiple codons can code one amino acid, but each codon only codes for one amino acid.

reading frames- correct groupings of codons

open reading frame- regular transcription mRNA with start and stop codons

template strand

strand used as a template in transcription

• template strand is determined by a specific nucleotide sequence

• template strand is always the same strand for a particular gene

• opposite strand further away may be used as a template strand for another gene

coding strand

non template strand that has identical code as codons (T in place of U)

Bacteria Transcription

transcription ends at “terminator”

transcription unit- the stretch of DNA being transcribed

RNA synthesis

Initiation- RNA polymerase II latches onto the promoter with the help of transcription factors. In eukaryotes, there’s a TATA box. The RNA polymerase II pries the two strands of DNA apart and RNA replication begins at the “start point” of the template strand

Elongation- The DNA strand being copied elongates

Termination- RNA polymerase II falls off after transcribing the polyadenylation signal, leaving behind a transcription unit

• a gene can be transcribed simultaneously by several RNA polymerases

• RNA polymerase doesn’t need a primer!

transcription initiation complex

compelete assembly of promoter, transcription factors, and RNA polymerase II

• in eukaryotes, there is a promoter called TATA box

RNA processing

alteration of mRNA before it is sent to the ribose

1. 5’ end gets a 5’ cap

2. 3’ end gets a poly A tail- polyadenylation: synthesized by poly A polymerase

functions

• facilitate the export of mRNA to the cytoplasm

• protects mRNA ftom hydrolytic enzymes

• helps ribosomes attach to the 5’ end

RNA splicing

removal of introns, and noncoding segments, by spliceosomes

spliceosomes- proteins and several small RNA that recognize splice sites

exons- translated into amino acids and expressed

Ribozymes

catalytic RNA molecules that can splice RNA

• properties that allow it to function as an enzyme

1. Able to form 3D structure because it can base pair by itself

2. Some RNA bases have functional groups that can catalyze

3. RNA can hydrogen bond with other nucleic acids

alternate RNA splicing

gene can give rise to multiple different polypeptides depending on which segments are considered as exons

domains

different regions of proteins

• different exons code for different domains

exon shuffling

mixing and matching of different exons of different genes that may result in new proteins

accurate translation requires two instances of molecular recognition

first: aminoacyl-tRNA synthetase correctly matches tRNA with amino acid

second: correct match between tRNA anticodon and mRNA codon

wobble

flexible base pairing of the third base allows some tRNAs to bind to more than one codon

Ribosomes

• made up of proteins and ribosomal RNAs

• eukaryotic ribosome is bigger than prokaryotic and has a different molecular composition- allowing antibiotics to inactivate bacterial ribosomes without hurting the eukaryotic one

• ribosomes facilitate the coupling of tRNA with mRNA codons

E site: where the tRNA leaves

P site: holds the polypeptide chain

A site: incoming tRNA and amino acid docking site

translation steps

initiation- small ribosomal subunit binds with mRNA and indicator tRNA that carries amino acid methionine. The small subunit moves along the mRNA until it reaches the start codon AUG. Initiation factors bring in the large subunit that completes the translation initiation complex.

elongation- amino acids are added one by one to the C terminus of the growing chain with the help of elongation factors

1. codon recognition- GTP required: anticodons on tRNA correctly bind to mRNA codons

2. peptide bond formation- rRNA of large ribosomal subunit forms polypeptide bonds between carboxyl ends of amino acids. The polypeptide chain is transferred from P site to A site tRNA.

3. translocation- GTP required: A site tRNA moves over to P site and P site tRNA exits through the E site.

Termination- stop codon is reached and release factor binds to site A and binds water instead of amino acid. The hydrolysis reaction causes the whole ribosomal subunit to fall apart.

targeting functional protein

free ribosome- in the cytosol and synthesizes proteins that function in the cytosol

bound ribosome- bound to the ER and synthesizes proteins that function in the endomembrane system or secreted

• synthesis starts and finishes in the cytosol unless polypeptide signals attach

signal peptide- attaches to polypeptide and signals it to go to the ER

signal recognition particle (SRP)- binds to signal peptide and escorts it to the ER

2. the signal peptide is removed by the enzyme

3. polypeptides are directed to other organelles

polyribosome/ polysome

enables a cell to make many polypeptide copies very quickly

• in prokaryotes, the whole process is even faster because transcription and translation can occur almost simultaneously

mutations

changes in the genetic information of the cell

point mutations- changes in one nucleottide PAIR of a gene

• single nucleotide pair substitutions

• nucleotide pair insertions or deletions

silent mutations

no effect on amino acid produced by a codon because of redundancy in the genetic code

missense mutations

still code for an amino acid, but not the correct amino acid

Nonsense mutations

change an amino acid codon into a stop codon; most lead to a nonfunctional protein

frameshift mutation

Insertion or deletion of nucleotides (not pairs) may alter the reading frame

mutagens

physical or chemical agents that can cause mutations

• Most carcinogens (cancer-causing chemicals) are mutagens, and most mutagens are carcinogenic

gene editing

altering genes in a specific way

• CRISPR-Cas9 protein Cas9 is guided by guide RNA that leads to a target gene. Cas9 will cut both ends of the segment that’ll trigger a DNA repair system where the repair enzymes will add or remove random nucleotides- disabling a given gene

• A template can be introduced with a normal copy of the gene that can be corrected