From gene to protein Flashcards
What is gene expression?
The process of going from DNA to a functional product
Gene expression diagram
Cytoplasm with nuclear envelope and nucleus.
Inside the nucleus; During transcription, DNA turns into Pre-RNA DUring RNA processing to mRNA; translation occurs; ribosome & polypeptide
DNA (deoxyribonucleic acid) is the heritable material
that is ….
used to store and transmit information from
generation to generation
RNA (ribonucleic acid) acts as a messenger to…
allow the information stored in the DNA to be used to
make proteins
Proteins carry out …
cellular functions
What are the 3 main steps in gene expression ?
Transcription of RNA from DNA
Processing of the Pre-mRNA script
Translation of the mRNA to a protein
What occurs during Transcription?
1- Polymearase binds to promoter
2- moves downstream through the gene, transcribing rna
3-detaches after terminator reached
RNA uses what diff base?
RNA uses nitrogenous base uracil instead of DNA which uses thymine
Is RNA double stranded like DNA
No- it is single stranded
3 main steps in transcription?
Initiation
Elongation
Termination
initiation is..
Assembly of multiple proteins required before transcription can commence
is 5-3 coding (non-template) or non coding (template) strand
5-3 is non-template strand
3-5 is template strand
WHich EUkaroytic promoter is used?
TATA box 25nt upstream
what are the 3 steps of initation
1- a eukaroytic promoter
2- several transcription factors bind to DNA- Assembly of several transcription factors including
the TATA box binding protein (TBP)
3- transcription intitation complex forms- RNA Pol II can now bind along with more transcription
factors to form the transcription initiation complex
How many nucleotides are exposed at a time when DNA unwound?
10-20 nucleotides exposed at a time when DNA unwound
What happens during Elongation:
Complementary RNA nucleotides added to 3’ end of
growing transcript (3’OH of transcript binds with 5’ phosphate of
incoming nucleotide)
it forms a phosphodiester bond
Double helix reforms as transcript leaves the template strand
WHat happens during termination
after transcription of the polyadenylation signal
(AAUAAA) nuclear enzymes release the pre-mRNA and RNA
polymerase then dissociates from the DNA
Fidelity (proofreading) is less than for DNA replication
WHat are the 3 steps in mRNA processing
capping, tailing, splicing
Capping:
a modified guanine nucleotide is
added to the 5’ end
Tailing:
50-250 adenine nucleotides (polyA)
are added to the 3’ end
Why do capping & tailing occur
Capping and tailing are thought to facilitate export,
confer stability and facilitate ribosome binding in cytoplasm)
splicing?
Introns are removed from the transcript
Exons:
regions that remain in mature RNA (includes UTR)
UTR:
untranslated regions at 5’ and 3’ ends of mRNA
Introns:
intervening regions that do not remain in mature RNA
Where does splicing occur?
At the spliceosome, within the nucleus
What is the spliceosome
Spliceosome: a large complex of proteins and small RNAs
What happens durinf splicing
Introns are removed from the transcript and exons
are rejoined to form mature mRNA
ALternative splicing
is a process by which different
combinations of exons are joined together. This
results in the production of multiple forms of mRNA
from the same pre-mRNA population.
Alternative splicing allows for multiple gene products from the same gene
~20,000 genes, there could be many times that number of proteins!!
protein sequence determines ….
its final structure
structure determines..
function
DNA mutations can affect …
ability of the protein to function
What happens in translation overview
Mature mRNA transcript exits nucleus and is bound by the ribosome
Codons are translated into amino acids
tRNA molecules within the cytosol with specific anticodons
carry corresponding amino acids
Hydrogen bonds form between mRNA and anticodon of the
appropriate tRNA
The amino acid is added via peptide bonds to the growing
polypeptide chain
Ribosome has binding sites ….
for mRNA and tRNA
Is the mrna binding site on the ribosomes small or large subunit
small
Ribosome
A site: holds “next-in-line” tRNA
P site: holds tRNA carrying the
growing polypeptide
E site: tRNAs exit from here
RNA is the physical link between …
the mRNA and the amino acid sequence of proteins
Transfer RNA (tRNAs) – the translatorObjective
Translation - initiation – a closer look
Initiator tRNA = tRNA carrying methionine (Met)
Small ribosomal subunit with initiator tRNA already bound binds 5’ cap of mRNA
Small ribosomal subunit scans downstream to find translation start site (AUG)
Large ribosomal subunit then binds
– completing the initiation complex
Energy (GTP- Guanosine triphosphate)
is required for assembly
Initiator tRNA = tRNA carrying methionine (Met)
Hydrogen bonds form between initiator anticodon and mRNA
translation - elongation – a closer look
Codon recognition:
base pairs with complementary anticodon
GTP invested to increase accuracy / efficiency
Peptide bond formation:
A large subunit rRNA catalyses peptide bond formation
Removes it from tRNA in P site
Translocation:
moves tRNA from A to P site
tRNA in P site moves to E and is released
Energy is required (GTP)
Empty tRNAs are ‘reloaded’ in the cytoplasm using aminoacyl-tRNA synthetases
translation - termination – a closer look
1- ribosome reaches a stop codon on mRNA
mRNA stop codon in the A site
is bound by a release factor
2- release factor promotes hydrolysis
Bond between p-site tRNA and
last amino acid is hydrolysed,
releasing polypeptide
3- ribosomal subunits and other components disssacoaite
Hydrolysis of two GTP molecules
required
Ribosome components can be recycled
Gene expression is tightly..
regulated
Multiple control points of gene expression..
Transcription factors need to assemble, and
DNA needs to be accessible
capping, extent of polyadenylation, alternate
splicing, producing an mRNA able to be translated
specific proteins assist in nuclear export of mRNA
regulatory proteins can block translation,
variable mRNA life-spans
Why is control of gene expression important?
to achieve the right thing at the
right time in the right place!!
(this is temporal and spatial control)
Housekeeping (commonly used) proteins are continuously produced
* protein and mRNA are present in large quantities (e.g. Tubulin)
* typically, have longer “half life” in cells
Other proteins are produced in response to stimuli as required
* cell signaling (e.g. ligand binding a cell surface receptor, or activating an intracellular receptor)
* signal transduced and may enter nucleus to activate transcription
* results in the production of a short-lived protein to carry out the required function
Amino acid properties
The side chains (R groups) determine
the properties of each amino acid
There are twenty standard (coded for) amino acids
WHich is N terminus & C terminus
N- 5
C-3
Primary strcuture
Protein sequence (primary structure)
is determined by DNA sequence
Held by covalent bonds between amino
acids (relatively strong)
The polypeptide starts to form secondary
structures as soon as it leaves the ribosome
Secondary structure
Held by weak hydrogen bonds to
form alpha helix and beta sheets
Tertiary structure
3D shape stabilized by
side chain interactions
Quatenary structure
Multiple proteins associate
together to form a functional
protein
Not all proteins form
quaternary structures
Protein processing and sorting
All translation commences on
free ribosomes
Many proteins are processed
and sorted through the RER
and Golgi – but not all
Proteins destined to function in
the cytosol – complete
translation on free ribosomes
Proteins that go through the
endomembrane system -
complete translation at fixed
ribosomes on the RER
Signal peptides direct ribosomes..
to RER
Signal peptide
at N terminus of the protein (~20aa)
SRP
signal recognition particle
A secretory protein such as insulin
is solubilized in lumen, while a..
membrane protein remains
anchored to the membrane
Both then go to the Golgi via
vesicles for further maturation
Post‐translational modifications
Translation is now complete, but the protein may not yet be functional
Common (there are 100s) post‐translational modifications include
Phosphorylation (addition of a phosphate group)
* Methylation (addition of a methyl group)
* Acetylation (addition of an acetyl group)
* Biotinylation (addition of biotin)
* Carboxylation (addition of a carboxylic acid group)
* Carbohydrate addition (particularly for membrane bound proteins, eg.
glycoproteins)
* Cleavage
* Ubiquitination
Can confer activity – eg. via phosphorylation or enzyme cleavage
or ability to interact with other molecules – eg. biotinylation, methylation of histones
or direct to particular locations – eg. ubiquitination for proteasome degradation
Some occur within the Golgi, others in the cytosol
modification errors could lead to non-functional proteins
