1.4 gene expression

gene

gene

a hereditable sequence of DNA that encodes for some RNA that has function

the basic unit of heredity

transcription

the process to produce a complimentary strand of RNA using the sequence in genes

translation

the process of linking amino acids to produce proteins using some RNAs from transcription (mRNA)

exons

sequences involved in coding for proteins in eurkaryote genes, short for expressed sequences

introns

non-coding sequences in eukaryotes, which are removed from mRNA prior to translation

short for intragenic sequences

central dogma

transcription: DNA → RNA

translation: RNA → protein

reverse transcription: RNA → DNA

RNA polymerase

enzyme responsible for producing an RNA transcript from DNA

reverse transcriptase

enzyme responsible for producing complementary DNA (cDNA) from mRNA, aka reverse transcription

DNA

• double stranded

• deoxyribose sugar

• bases are AGCT

• stable in alkaline conditions

• encodes genetic information

• meant to be stable

RNA

• single stranded for base pairing

• ribose sugar

• bases are AGCU

• susceptible to alkaline hydrolysis

• translated into proteins or for particular functions

• temporary (degraded after use)

  • mRNA (messenger), tRNA (transfer), rRNA (ribosomal)

protein functions

over half of a cell’s dry weight

functions include:

• structural components

• tensile strengthening

• immunity and clotting

• cytoskeleton

• muscle contraction

• cellular transport

• transport of nutrients and gases

• catalysis

• hormones

• organization

• cell adhesion

• regulation

amino acid

compounds with an amino group (basic) and a carboxyl group (acidic) attached to a central carbon

an R group distinguishes individual traits, also attached to central carbon

protein formation

formed through dehydration synthesis (condensation) where a water molecule is removed to form a peptide bond/amide linkage between carboxyl group of the original amino acid and the amino group of an adjacent amino acid

two amino acids → water + dipeptide

primary structure

amino acid sequence (chain)

• important for structural functions of the protein

secondary structure

initial repetitive structure formed from hydrogen bonding between amino groups and carboxyl groups

• alpha helices - hydrogen bonds within sequence

• beta pleated sheets - hydrogen bonds between sequences

• important for structural functions of the protein

tertiary sequence

folding of the polypeptide chain into 3D shapes

• stabilized by covalent bonding of R group interactions (disulfide bridges) and hydrophobic/philic interactions

• important for functional proteins

quaternary structure

interaction between multiple polypeptides in tertiary structure

• stabilized by hydrophobic/philic interactions

• important for functional proteins

protein synthesis: transcription

mRNA transcript is synthesized using a DNA template by RNA polymerase

1. RNA polymerase is directed to a gene by association with transcription factor(s), which direct RNA polymerase to a promoter (regulatory sequence)

2. when RNA polymerase and transcription factor(s) bind to the promoter, a transcription bubble is created

3. the 3’-5’ strand of DNA is used as a template to create a complementary mRNA molecule - called the antisense strand

4. mRNA synthesis proceeds 5’-3’, using the 3’-5’ antisense strand as a template

5. synthesized mRNA strand matches the DNA ‘sense’ strand which ‘codes’ for amino acid sequence in translation

6. complimentary free NTPs to the antisense strand are assembled by RNA polymerase, where uracil replaces thymine

   1. energy for this comes from breaking a phosphate bond to release pyrophosphate

• occurs in the nucleus in eukaryotes

promoter (transcription)

a regulatory, non-coding sequence upstream (-5’) of the gene

how is a transcription bubble formed? (transcription)

breaking the hydrogen bonds between complimentary strands when the RNA polymerase and transcription factors bind onto the promoter

antisense strand

coding strand (RNA transcript)

pre-mRNA transcript processing

eukaryotic genes contain exons and introns (expressed and intragenic sequences)

introns must be removed before translation

splicing

process to remove introns by bringing the 3’ end of exons to the 5’ end of adjacent exons, excising out the intron

• mediated by small nuclear ribonucleoprotein (snRNPs) complex called a spliceosome

  • assemble at the intron and cause it to ‘loop out’ to bring its two ends together

• can produce different proteins through alternative splicing (some exons can be spliced out to create different transcripts)

• a methylated guanosine cap is added to the 5’ end of RNA transcript

• a poly-A tail is added to the 3’ end

  • the cap and tail protect the transcript from degradation by exonucleases in the cytoplasm and aid in exporting mature mRNA transcript and promote translation of mRNA

protein synthesis: translation

occurs on ribosomes

1. initiation - the ribosome assembles around the mRNA and the first tRNA attaches to the start codon

2. elongation - tRNA transfers associated amino acid to the tRNA corresponding to the next codon. it is released and the process is continued to synthesize the peptide

3. termination - once a stop codon is encountered the ribosome disassembles and releases the completed polypeptide

ribosomes

ribonucleoproteins composed of rRNA and proteins

composed of two subunits

• small subunit that binds onto mRNA and subunit

• big subunit that binds onto tRNA and its amino acids and small subunit

prokaryotes have 70S ribosomes consisting of 30S and 50S subunits

eukaryotes have 80S ribosomes consisting of 40S and 60S subunits

large subunit ribosome binding sites

three sites for tRNA, can bind to two tRNA at a time

• E (exit)

• P (peptidyl)

• A (aminoacyl)

codon

a triplet of bases, mRNA sequence is read as a codon

bound by tRNAs through an anticodon (a complementary triplet base pairing)

each codon encodes for a specific amino acid that the specific tRNA will carry

64 possible codons for 20 amino acids

• there is some redundancy - multiple codons can code for the same amino acid

start codon (AUG)

AUG - used to signal the start of translation

• encodes for methionine (Met)

• in prokaryotes, a formyl group is added → N-formylmethionine (fMet)

nonsense codon / stop codon

end the translation

• codons which have no associated amino acids

• UAA, UGA, UAG

aminoacyl-tRNA synthetase

tRNA activating enzymes specific to one of the 20 amino acids and correct tRNA molecule

• amino acids are attached to tRNAs

• attachment of an amino acid to the active site, hydrolysis of ATP releases a pyrophosphate and covalently bonds remaining AMP to the amino acid to activate it

• attachment of tRNA to the other active site of tRNA activating enzyme causes the activated amino acid to attach to the tRNA

• releases AMP

translation: initiation

1. mRNA binds onto the small ribosomal subunit at mRNA binding site

2. initiator tRNA carrying methione binds to the start codon (AUG)

3. the large ribosomal subunit assembles onto the small one with initator tRNA at the P site

4. another tRNA carrying an amino acid for the next codon sequence binds onto the mRNA sequence, occupying the A site

5. when P and A sites are both occupied, a peptide bond is formed between amino acids, freeing them from the tRNA at P

6. the growing peptide is only associated with the tRNA at the A site

translation: elongation

1. the ribosome translocates along the mRNA so that the tRNA at the A site moves to the P site

2. the A site is now free for another amino acyl tRNA that matches the next codon sequence

3. the tRNA at the P site moves to the E site where it is ejected from the ribosome

4. at the P site, the amino acid is attached to the peptide chain and is freed from the tRNA

5. this continues until a stop codon causes translation to stall and the ribosome to disassemble

results in termination of translation and release of completed polypeptide

ribosomes on ER

produce proteins for use in the ER, golgi apparatus, lysosomes, plasma membrane, or for secretion outside of the cell

free floating ribosomes

make proteins for use in the cytoplasm, mitochondria, or chloroplasts

signal recognition protein

binds onto the first part of the polypeptide as it is being synthesized by translation

• halts translation until the ribosome can bind to a receptor of the ER

• translation continues once bound to the ER, with the growing polypeptide moving into the lumen of the ER

prokaryotic protein synthesis

prokaryotes do not have a membrane bound nucleus and do not have introns

• mRNA does not have to be processed

• transcription and translation are coupled - occur simultaneously

• multiple ribosomes can bind to an actively growing transcript

gene expression regulation

sequences within the gene (upstream or downstream) may exist that serve as binding sites for proteins

• either enhance (enhancers) or repress (silencers) transcription

• e.g. activator protein binding to an enhancer site can increase the likelihood of RNA polyermase binding to the gene’s promoter

• e.g. repressor protein binding to the silencer site can physically block RNA polymerase from transcribing the gene from hte promoter

gene regulation occurs through the interaction of diffusible protein regulators with DNA requences

operon

the organization whereby a cluster of genes are controlled by one promoter (in prokaryotes)

• allows prokaryotes to react quickly to changing conditions - genes relating to some function can all be expressed and turned on together

lac operon

allows the metabolism of lactose

• consists of three genes controlled by a single promoter:

  • lacZ - encodes for beta-galactosidase which cleaves lactose into glucose and galactose

  • lacY - encodes for a beta-galactoside permease that allows lactose into the cell

  • lacA - encodes for a galactoside acetyltransferase that attaches an acetyl group from acetyl CoA to beta-galactoside

• downstream from the lac promoter is a binding site for the lac repressor protein

• upstream of the promoter is a binding site for a Catabolite Activator Protein (CAP)

lac operations when glucose is present

• cyclic adenosine monophosphate levels are low

• bacterial cell uses this sugar as it is more easily metabolized through glycolysis

• lac repressor is constantly produced at a low level (constitutively) and binds to the repressor site, preventing RNA polymerase from transcribing the operon

• if lactose is present it can bind to the repressor protein and decrease its affinity for repressor site to allow transcription

lac operations in absence of glucose

• cyclic adenosine monophosphate (cAMP) levels are high

• cAMP can bind to CAP which can then bindo onto the activator site

• binding of cAMP-CAP promotes RNA polymerase to transcribe the lac operon

when is lac operon transcription maximized?

in the presence of lactose - to remove the lac repressor

in the absence of glucose - to enhance RNA polymerase binding

eukaryotic DNA and histones

highly organized and condensed into chromosomes

• associated with alkaline proteins (histones) that act as spools which the DNA winds around

• basic nature of histones allows its attachment to the acidic phosphate backbone of DNA

histone subunits and nucleosome

H1, H2a, H2b, H3, H4

• H2a, H2b, H3, and H4 form the core around which the DNA wraps around

• H1 acts as a linker that anchors the core onto the DNA

• the core is an octamer made of two tetramers, each made of a dimer of H2a and H2b and a dimer of H3 and H4

structural unit is called a nucleosome

euchromatin

transcriptionally active DNA

heterochromatin

transcriptionally inactive DNA

histones in gene regulation

• acetylation (adding a CH3COO-) on amino terminal ends of lysine residues can neutralize the positive cjarge allow the histone to be more relaxed

  • this opens up the DNA to allow for transcription factors and enhance gene activity

autoradiography

using tritiated uracil to identify regions of active transcription in uncoiled chromosomes (euchromatin)

allows for a more accurate estimation of the length of the chromosome because they are uncoiled