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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
RNA polymerase is directed to a gene by association with transcription factor(s), which direct RNA polymerase to a promoter (regulatory sequence)
when RNA polymerase and transcription factor(s) bind to the promoter, a transcription bubble is created
the 3’-5’ strand of DNA is used as a template to create a complementary mRNA molecule - called the antisense strand
mRNA synthesis proceeds 5’-3’, using the 3’-5’ antisense strand as a template
synthesized mRNA strand matches the DNA ‘sense’ strand which ‘codes’ for amino acid sequence in translation
complimentary free NTPs to the antisense strand are assembled by RNA polymerase, where uracil replaces thymine
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
initiation - the ribosome assembles around the mRNA and the first tRNA attaches to the start codon
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
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
mRNA binds onto the small ribosomal subunit at mRNA binding site
initiator tRNA carrying methione binds to the start codon (AUG)
the large ribosomal subunit assembles onto the small one with initator tRNA at the P site
another tRNA carrying an amino acid for the next codon sequence binds onto the mRNA sequence, occupying the A site
when P and A sites are both occupied, a peptide bond is formed between amino acids, freeing them from the tRNA at P
the growing peptide is only associated with the tRNA at the A site
translation: elongation
the ribosome translocates along the mRNA so that the tRNA at the A site moves to the P site
the A site is now free for another amino acyl tRNA that matches the next codon sequence
the tRNA at the P site moves to the E site where it is ejected from the ribosome
at the P site, the amino acid is attached to the peptide chain and is freed from the tRNA
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