WEEK 2 - RIBOSOMES, PROTEIN SYNTHESIS, ENDOPLASMIC R. Flashcards
RIBOSOMES, PROTEIN SYNTHESIS, ENDOPLASMIC RETICULUM
ribosomes
- All living cells - role in protein synthesis
- Two main subunits, comprised of RNA and
proteins - Small subunit 40s– decodes genetic information from mRNA
- Large subunit 60s-catalyses formation of
peptide bonds between amino acids to form a polypeptide chain. - large and small ribosome subunits are assembled in the nucleolus, where newly transcribed and modified rRNAs are brought into association with the ribosomal proteins that have been transported into the nucleus after their synthesis in the cytoplasm
- Ribosome - highly ordered, with rRNA molecules forming a scaffold that positions the proteins in the correct orientation to carry out their functions
why do ribosome sizes have s e.g. 40s, 60s
- to do with their sedimentation coefficient
- when you separate them out, when you look at them biologically, you can you can basically sediment the different proteins.
- s stands for Svedberg unit
- measure of how fast a substance sediments during centrifugation
more about ribosomes
- rRNA molecules direct the catalytic steps of protein
synthesis - Active cells (rapidly growing = high protein synthesis) – more ribosomes
- Ribosomes are complexes of rRNA molecules and
proteins - Although only a few rRNA molecules are present in
each ribosome, these make up ~ 50% ribosomal mass - The remaining mass consists of a over 80 different proteins
- To maintain correct reading frame (codon) and ensure accuracy (1 error every 10,000 aa) protein
- Takes ~1 minute to synthesise a protein
ribosomes - roles of the subunits
- when subunits are not in use they are separate, large and small separate and join together only when we get a messenger RNA molecule (mRNA)
- Two subunits join on mRNA near 5’ end
- Ribosome has 4 binding sites for RNA – one for mRNA and three (A,P,E) for tRNA
- mRNA pulled through ribosome – 3 nucleotide sections at a time
- tRNA molecule is held tightly at the A and P sites only if its anti-codon form base pairs with a complementary codon (allowing it to wobble)
- tRNAs - adaptors to add each amino acid in the correct sequence
- Stop codon – two subunits separate again
- 4 aa per second (bacteria faster)
translating mRNA
- ribosome subunits come together, tRNA binds and a growing polypeptide chain which the A site is getting the chain to move down
- carboxyl end of the polypeptide chain is released from the tRNA at the P site and joined to the free amino group of the aa linked to the tRNA at A site, forming a new peptide bond
- eventually we don’t need the tRNA anymore since that’s what was localising the bases in the right position so is ejected
- large and small ribosome subunit translocate resulting in the entire ribosome to three nucleotides along the mRNA
- new tRNA is inserted and shortly after its bound it is also ejected
protein synthesis translation - key points
- Translation - in cytosol on ribosome and process by which protein is created
- Each amino acid attached to tRNA
- Nucleotides read in sets of three
- Initiated via a start codon recognised by initiator tRNA
- Reaction driven by elongation factors, using
GTP hydrolysis - Process continues until it reaches a stop codon
- Release factor binds to the ribosome – terminates translation and polypeptide is released
- Folding of newly synthesised proteins assisted by chaperone proteins
- Control mechanisms to destroy incorrectly folded proteins, since it could destroy the cell or lead to uncontrolled cell growth
DNA replication
- semi-conservative replication
- most genes contain information to make
proteins. - For minority of genes, final product is the RNA
molecule itself – generally encoded by RNA
polymerase I or III.
protein synthesis
Protein Synthesis – 2 stages:
* Transcription –DNA copied into strand of
mRNA
* Translation –formation of a chain of amino
acids
- DNA is transcribed into mRNA in the nucleus
- The mRNA leaves the nucleus and enters the cytoplasm
- The protein is translated from the mRNA sequence using the tRNA and amino acid
From DNA to RNA
- Many identical RNA copies can be made
from the same gene - Genes can be transcribed and translated with different efficiencies – different amounts of proteins so can make one or many
TRANSCRIPTION AND TRANSLATION –
KEY POINTS
- Nucleotide sequence “spells out” sequence of amino acids in a protein
- Properties/function of protein determined by 3D structure – depends
on linear sequence of the amino acids - 4-letter alphabet of DNA translates to 20-letter amino acid alphabet
Transcription
- DNA transcription - single-strand RNA molecule
complementary to one strand of the DNA double helix - Sequence of bases in RNA molecule - same as sequence of bases in the non-template DNA strand, except that a U replaces every T base in the DNA
Transcription - content
- DNA unzips: enzymes split apart base pairs and unwind the DNA
double helix - Bases pair up: Free nucleotides attach to complementary bases
along the new strands using RNA polymerase
New backbone formed: The sugar-phosphate backbone is assembled to complete the RNA strand
Three classes of RNA
- Messenger RNA (mRNA) - carry the coding sequences for protein synthesis and are called transcripts – carries message from DNA to cytoplasm
- Ribosomal RNA (rRNA) - forms the core of cell’s
ribosomes - Transfer RNA (tRNA) - carry amino acids to the ribosomes during protein synthesis
In eukaryotic cells, each class of RNA has its own polymerase - polymerase I, II, and III
RNA molcules
- single stranded
- Sugar – ribose
- Contain (U)racil not (T)hymine
- don’t form helices - fold into complex structures - stabilised by internal complementary base-
pairing
TRANSCRIPTION – COMPLETION AND
EDITING
- Once termination complete, mRNA molecule peels away from DNA template
- A nucleotide is added to 5’ end – capping (an N7-methylated guanosine)
- Noncoding nucleotide sequences (introns),
removed from mRNA strand – splicing - Sequence of adenine nucleotides called
a poly-A tail added to the 3’ end of the mRNA
molecule – polyadenylation - Poly-A tail signals that mRNA ready to leave
the nucleus – enters cytoplasm
mRNA
- Most variable class of RNA
- RNA polymerase II synthesises mRNA – requires transcription factors, signal, to initiate transcription
- mRNA cap - highly methylated modification of the 5′ end of RNA pol II-transcribed RNA.
- Protects RNA from degradation
- Recruits complexes involved in RNA processing, export and translation initiation
- Marks cellular mRNA as “self” - avoid recognition by innate immune system
- Many different mRNA molecules in a cell at any given time - some mRNA abundant, others rare
- Variable life-span
- transcripts for signalling proteins degraded in <10 mins
- transcripts for structural proteins may remain intact for >10 hrs
- Transcriptome - spectrum of mRNA molecules in a cell
- Each cell carries same DNA but transcriptome varies according to cell type and function.
* E.g. insulin-producing cells of the pancreas contain transcripts for insulin, but bone cells do
not. Even though bone cells carry the gene for insulin, this gene is not transcribed.
how is transcription regulated
- Initiation
RNA polymerase and transcription factors bind to the DNA strand at a specific
area that facilitates transcription - promoter region.
Promoter Region often includes specialised nucleotide sequence, TATAAA, (aka - TATA box) - Elongation
RNA polymerase moves down the DNA template strand in 3’ to 5’ direction,
adding complimentary nucleotides.
Remember – complimentary base pairing - Termination and editing
Elongation process needs to end and mRNA to separate from DNA template -
termination. Termination can occur as soon as the polymerase reaches the termination sequence, but in some cases a termination factor (protein) is also needed
translation
Every amino acid is represented by a three-
nucleotide sequence (codon) along the mRNA
molecule
e.g. AGC codon for serine, and UAA is a signal to
stop translating a protein (stop codon).
transcription content
- Each 3-letter combination “codes” for an amino acid
- An RNA sequence can be translated in any one of three different reading frames, depending on where the decoding process begins
- BUT, only one of the three possible reading encodes the required protein
transfer DNA (tRNA)
*nTranslation of mRNA into protein - adaptor
molecules that recognise and bind codon
and amino acid - tRNA
* Each tRNA ~ 80 nucleotides long
* Four short segments of each folded tRNA are
double-helical, producing a molecule - a cloverleaf
* The cloverleaf - further folding to form a
compact L-shape held together by hydrogen
bonds between different regions of the
molecule
* tRNAs contain some unusual bases -
produced by chemical modification after
the tRNA has been synthesized.
* e.g. bases Ψ (pseudouridine) and D
(dihydrouridine) are derived from uracil.
* The anticodon is the sequence of three
nucleotides that base-pairs with a codon
in mRNA
tRNA summary
- Matches amino acids with the appropriate codons in mRNA, pulls everything together
- Has two distinct ends - one binds to a specific amino acid, and the other binds
to the corresponding mRNA codon - During translation, tRNAs carry amino acids to the ribosome and join with their
complementary codons - Assembled amino acids are joined together as the ribosome, with its and
RNAs, move along the mRNA molecule in a ratchet-like motion - Resulting protein chains can be hundreds of amino acids in length – energy
dependent
tRNAs are covalently modified before they exit from the nucleus
- tRNAs are covalently modified before they are allowed to exit the nucleus by RNA polymerase III
- some tRNA precursors contain introns that must be spliced
HOW DOES A tRNA MOLECULE LINK TO THE CORRECT AMINO ACID?
- correct aa depends on enzymes called aminoacyl-tRNA-synthetases which covalently couple each aa to its appropriate set of tRNA molecules
- carboxyl end of the amino acid forms an ester bond to ribose – bc the hydrolysis of this ester bond is associated with a large favourable change in free energy an aa held in this way is said to be activated
- Aa linked to nucleotide at 3’ end of tRNA
- Two classes of synthetase – catalyses reaction so one links aa to 3’OH of ribose, other links to 2’-OH – esterification shifts as to 3’ position
- one enzyme for each aa
how amino acids join together
- formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming aa
- protein is synthesised from its N-terminal end to its C-terminal end, one aa at a time
- growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule.
- each addition disrupts this high-end covalent linkage but immediately replaces it with an identical linkage on the most recently added aa
redundancy
- Some amino acids have more than one tRNA, - some tRNAs are constructed to require accurate base- pairing only at the first two positions of the codon and can tolerate a mismatch (or wobble) at the third position
- This wobble base-pairing - why alternative codons for an amino acid differ only in their third nucleotide.
- some tRNA molecules can base-pair with more than one codon
- Humans have nearly 500 tRNA genes that encode tRNAs with 48 different anticodons
RNA polymerases
The 3 types of RNA polymerases (enzymes that polymerize nucleotides to make ribonucleic acid) are RNA Polymerase I, II, and III. RNA Pol I makes rRNA in the nucleolus; RNA Pol II makes mRNA, miRNA, and snRNA; and RNA Pol III makes tRNA and 5S rRNA.
RNAs Are Modified Before They Exit from the nucleus
- Eukaryotic tRNAs synthesized by RNA polymerase III
- tRNAs generally synthesised as larger precursor tRNAs, then modified mature tRNA
- Some tRNA precursors contain introns that must be spliced out - a cut-and-paste mechanism catalysed by endonucleases
- Trimming and splicing require precursor tRNA to be correctly folded in its cloverleaf configuration – quality control process
- Those that do not pass the tests are degraded by the nuclear exosome.
editing
proofreading stage
2 mechanisms –
* Favourable binding - Correct AA has
highest affinity for active side pocket of
synthetase (an enzyme that catalyzes the joining of two molecules, a process known as synthesis) and is therefore favoured.
correct aa it doesn’t bind to binding site, incorrect aa will bind and is recognised as incorrect and will be removed. There is an editing site which the synthetase tries to force/place the amino acid into. The dimensions of the editing site mean that the correct amino acid will not bind, but incorrect ones do (for similar aa to correct).
* Hydrolytic editing – is a proof reading mechanism in transcription which removes errors in RNA via the enzyme DNA polymerase. here error removal depends on the wrong nucleotide misfitting with the DNA template. when tRNA binds, synthetase tries to force adenylated AA into a second editing pocket
Dimensions EXCLUDE correct AA but allows
closely associated Aas. AA is removed from
the AMP by hydrolysis
Where does protein synthesis start
- Protein synthesis starts is important – errors could result in misreading/nonfunctioning
- Starts at AUG
- Initiator tRNA always carries formylmethionine – all newly made proteins have methionine as the first aa at the N-terminus
- Initiator tRNA-methionine complex loaded into small ribosomal subunit along with protein – eukaryotic initiation factors (eifs)
- Met-tRNA is capable of tightly binding the small ribosomal subunit without the complete ribosome being present and binds directly to the P-site
- Small ribosomal subunit then binds 5’ end of mRNA molecule – moves along mRNA to find AUG
- Initiation factors dissociate – allowing large ribosomal subunit to assemble with complex and complete ribosome
- Initiator tRNA remains at P site, A site vacant and protein synthesis begins
when does protein synthesis stop
- End of coding – one of three stop codons (UAA, UAG, UGA)
- proteins known as release factors bind to ribosome with stop codon at A site – forces peptidyl transferase to add water molecule instead of aa
- Frees carboxyl end of pp chain – chain is
released into cytoplasm - Ribosome releases bound mRNA molecule and separates into subunits
- New round of synthesis
proteins are made on polyribosomes
- generally proteins are thought of as being created one by one on a template, but we can have a situation where the polypeptide chain is synthesised one after the other
- Protein synthesis – 20 secs and several mins
- Multiple initiations
- As soon as preceding ribosome is out of the way the 5’ end of the mRNA is threaded into a new ribosome
- Most mRNA molecules esp being translated at high rates found as polyribosomes
- Polyribosomes ~80 nucleotides apart
control mechanisms
- Cell has backup measures to prevent
truncated (shorten or cutoff) or aberrant protein production. - several backup measures to prevent translation of damaged mRNA
- Broken mRNA – missing either 5’ cap or poly A tail – translation initiation won’t begin
- Nonsense mediated mRNA decay – prevents mRNA escaping from nuclear envelope, done when mRNA determined to have a nonsense or stop codon in wrong place e.g due to splicing. Happens as mRNA transported to cytosol
cellular rubbish bin : proteosome
- Removing unwanted and faulty
proteins is essential to “normal cell
function” therefore the cell has
developed a way to do this using the
proteosome - proteins enter through the top and has a cylinder type effect and will degrade the protein and end up with fragments coming out the bottom
proteosomes
- Proteosomes – abundant – 1% total proteins in cells.
- Dispersed in cytosol & nucleus
Destroys aberrant proteins in ER
ER detects protein, retrotranslocates to cytosol for destruction - complex cap selectively binds proteins that are marked by ubiquitin
- Structure – central hollow cylinder formed from multiple protein subunits that assemble a stack of four heptameric rings
- Contains six subunit protein ring – target proteins threaded into core - degraded
useful protein
- To be useful, protein needs to fold to be 3D conformation, bind any co-factors and any other modifications (non covalent interactions drive these changes)
- During folding the protein buries, hydrophobic residues in interior core.
Non-covalent bonding between parts of the molecule - covalent modification by glycosytation, phosphorylation, acetylation, etc.
- binding to other protein subunits
- Some proteins fold in exit tunnel of ribosome – simple structures only e.g. α
helices but major part of folding as protein emerges from ribosome exit tunnel
chaperones - Hsp70
- generally two types of chaperones (Hsp70 + Hsp60) that are heat shock proteins, key role in how proteins are formed inside cells and key to the right folding and formation
- Most proteins require molecular chaperones to fold properly or proteins may become ‘kinetically trapped’
- Many ways to fold unfolded or partially folded proteins – chaperones ensure correct pathway followed
- Many chaperones are heat-shock proteins (hsp) – synthesised in increased amounts when cells exposed to higher temperatures but constitutively expressed.
- Several major families of hsp incl. hsp60 & 70 – function in different organelles e.g. mitochondria contain own hsp60 & hsp70 different from those in cytosol and ER
- hsp70 ensures proteins fold correctly by rapidly releasing and binding short sequences through ribosome exit tunnel
- short stretches of hydrophobic aa that are abnormally exposed in misfiled proteins trigger the hydrolysis of ATP to ADP causing hsp70 to close down
- it clamps down on it, delaying the folding of the emerging protein until enough of it has been made to begin to fold correctly
folding - Hsp60
- some proteins cannot fold with HSp70 alone
- Hsp60 forms barrel shape - isolation chamber.
- a misfolded protein is initially captured by hydrophobic interactions with the exposed surface of the opening, intial binding often helps to unfold and misfiled proteins
- subsequent binding of ATP and a cap releases the substrate in an inclosed space
- twisting of the chamber subunits causing protein to fold with hydrophobic and hydrophilic interactions
- binding of additional ATP molecule ejects the cap and the protein is released and the other half the symmetrical barrel operates at a time
- Known as a chaperonin
regulated destruction of proteins
Why eliminate proteins?
Misfolded or abnormal or Damaged
Alteration of cell state – specific lifetime of normal proteins
How?
Many proteins contain short unfolded regions
Addition of ubiquitin
General mechanisms –
* ubiquitin will bind to lysine
* activation of ubiquitin ligase
* activation of degradation signal
* Creation of a ubiquitinylation site in response to intracellular or extracellular signals
* additional ubiquitin molecules can be added to create polyubiquitin chain which can deliver the protein to a complex protease known as proteasome
in summary - protein synthesis
- initiate transcription
- capping, elongation, splicing process
- cleavage, polyadenylation and termination
- export
- leaves nucleus and initiation of protein synthesis (translation) and mRNA degradation
- completion of protein synthesis and protein folding
- protein degradation or complete protein
signalling - how do proteins get to their final destination
- What destination?
Secretion, integration in plasma membrane, inclusion in lysosomes - Same initial first few steps of pathway starting in ER
- Proteins for mitochondria, chloroplast or nucleus – 3 separate mechanisms to direct where to go
Proteins for cytosol remain where synthesied - Signal sequence key to process –
Directs protein to location in cell and are recognised as a short sequence of aa
Removed during transport or after protein reaches final destination - Proteins no longer needed – degraded – signal embedded in protein structure
signalling - signal recognition particles (SRP)
- binds an ER signal sequence on a partially synthesised polypeptide chain and directs the polypeptide and its attached ribosome to the ER
- Recognises one class of signal sequence
- Binds signal sequence on ribosome – transfers entire ribosome and incomplete polypeptide to ER
- PP + signal sequence moved into ER lumen when synthesised.
In lumen – many glycosylated (the attachment of carbohydrates to the backbone of a protein through an enzymatic reaction) and moved to Golgi complex, sorted and sent to lysosomes, the plasma
membrane, or transport vesicles. (see Dr. Bailey’s lectures) - Proteins targeted to the nucleus have an internal signal sequence that is not cleaved once the protein is successfully targeted.
Proteins targeted to mitochondria and chloroplasts in eukaryotic cells use an amino-terminal signal sequence. - Some eukaryotic cells import proteins by receptor-mediated endocytosis (may be absorbed by organelle)
- All cells eventually degrade proteins, using specializes proteolytic systems.
Signal sequencing - targeting proteins to the ER
- Signal sequence - translocation into the
lumen of the ER - The carboxyl terminus - a cleavage site,
where protease action removes the
sequence after the protein is imported
into the ER - Signal sequences vary in length (13 to 36)
aa residues but have - ~ 10 to 15 hydrophobic amino acid
residues - one or more positively charged
residues - near the amino terminus - a short sequence at the carboxyl
terminus (relatively polar), typically
residues with short side chains (especially
Ala) at positions closest to the cleavage
site - initiation of protein synthesis
- signal sequence appears early in the synthetic process (at amino terminus)
- signal sequence and the ribosome bound by the SRP
- SRP binds to GTP (signal sequence) and halts elongation of the polypeptide/ temporary halts protein translation- 70 aa long and signal sequence has completed
- SRP directs the ribosome and the incomplete polypeptide to SRP receptors in the cytosolic face of the ER lumen; the newly formed polypeptide is delivered to a protein translocation complex in the ER which interacts directly with the ribosome
- SRP dissociates from the ribosome and hydrolysis of GTP in SRP and SRP receptors
- elongation (protein synthesis) of polypeptide now resumes
- signal sequence removed by signal peptidase within ER lumen
N-linked glycosylation
- Further modification of newly synthesised proteins..
- Attachment of an oligosaccharide (carbohydrate consisting of several sugar molecules) to N (amide nitrogen of
asparagine residue (Asn)) – also known as
N-Glycosylation - Linkage important for structure and function of eukaryotic proteins.
- Sequence of events…
- Removal of signal sequence – polypeptide folding, disulfide bonds formed
- Linkage to oligosaccharides – through Asn residues
- 14 residue core oligo built up – on cytosolic face of membrane and then luminal. This then transferred from dolichol phosphate donor molecule to Asn residues.
- After transfer – core oligo trimmed and adapted
glycosylation
- Modified proteins moved to intracellular
destinations - Proteins travel from the ER to the Golgi complex in transport vesicles – see Dr. Bailey’s lectures
- core oligosaccharide is built up
- the first steps occur on the cytosolic face of the ER
- Translocation moves incomplete oligosaccharide across the membrane
- completion of oligosaccharide in lumen of ER
- core oligosaccharide transfered from dolichol phosphate to an Asn residue of the protein
- protein synthesis continues - further modified in the ER and the Golgi complex
- sugar residues are retained in the final structure of all N-linked oligosaccharides
- phosphate is hydrolytically removed to
regenerate dolichol phosphate - released dolichol pyrophosphate is again translocated so that the pyrophosphate is on the cytosolic face of the ER
the nucleolus
- Discrete area within the nucleus of eukaryotic cell
- Site for synthesis and processing of rRNA and production/assembly of ribosomes
- Not bound by a membrane – condensate of macromolecules
- Size/colour of nucleolus ∝ activity of the cell - large nucleolus (or multiple nucleoli) suggests cell is synthesising a large amount of protein
- The regions of the chromosomes that contain clusters of genes encoding ribosomal RNA loop to the nucleolus
endoplasmic reticulum
rough- ribosomes and Synthesizes proteins, which are then folded, checked for quality, and sent to other parts of the cell
smooth- Synthesizes and stores lipids, and helps detoxify the cell.
ER is structurally and functionally diverse
- Distinct regions of the ER become highly
specialised. - Entails changes in the proportional abundance of different parts of the ER
- Rough ER – cisternae (flat sheets studded with ribosomes)
Smooth ER – connected via tubules - Tubules and sacs interconnect, and membrane is continuous with the outer nuclear membrane
endoplasmic reticulum content
- Membrane of the endoplasmic reticulum (ER) >50% total membrane of an average animal cell
- Membrane system encloses a single internal space - ER lumen, which is continuous with the space between the
inner and outer nuclear membranes - The ER often occupies more than 10% of the total cell volume
function of ER
- Central role - biosynthesis of both lipids and proteins, major portion of the cell’s protein synthesis occurs on the cytosolic surface of the rough ER
- Rough – lysosomal proteins (digestive enzymes), membrane proteins, proteins for export from cell
- Smooth – make and store proteins, carbohydrates, phospholipids & steroids, detoxification
- in ER lumen - protein folding, oligomerisation, formation of disulphide bonds, N-linked oligosaccharides added (post-translational modification)
- Pattern of N-linked glycosylation indicated extent of protein folding - proteins leave the ER only when they are properly folded
ER - where some proteins are made
- Ribosomes - required for translation
- Ribosome recruited to the ER membrane
through a lipophilic sequence in the
polypeptide chain being produced - PP chain moves into the ER (no ATP)
- no difference between translation in cytoplasm
and ER in terms of energy use - The protein may be released into the ER
lumen or integrated into the membrane
ROUGH VS SMOOTH
Rough ER
* Most secreted proteins are synthesised by the ribosomes
* Cells specialised to secrete large amounts of protein are packed with rough ER. e.g exocrine cells of the pancreas (rough ER makes up 60% of these cells’ membranes)
Smooth ER
* Transitional ER - transport vesicles carrying newly synthesised Functions for the smooth ER are more diverse and can become highly specialised.
* transitional er contains exit sites, where the transport vesicles that contain newly synthesised lipids and proteins are made and will be released into the Golgi
* Proteins and lipids bud off for transport to the Golgi apparatus.
* Cells that synthesise steroid hormones contain prominent smooth ER to accommodate the enzymes that make cholesterol and modify it to form a variety of steroid hormones
* Membranes contain enzymes that catalyse detoxification of drugs and various harmful compounds produced by metabolism e.g. cyt P450
* Sequesters Ca2+ from the cytosol - muscle cells (smooth ER = sarcoplasmic reticulum). Release and reuptake of Ca2+ by the SR triggers myofibril contraction and relaxation of muscle
mechanism
- Translation of eukaryotic mRNA starts on
free ribosomes in cytosol. - If protein destined for lysosomes,
secretion or cell membrane – directed to
ER by co-translational localisation. - When N-terminus emerges from
ribosome, signal sequence for
localisation to RER recognised by Signal
recognition particle. - Complex translated to RER to lumen
what is meant by RNA processing and what is the point of it (why don’t we just have multiple genes)
in this case - one original RNA template from one gene, which is then chopped up into the 3 rRNAs.
In one place the polymerase doesn’t need to find 3 promoter regions, just hops on and does it all in one go. Enables many more polymerases to be on the same gene (overall more efficient)
what are the three steps of translation?
codon recognition, peptide bond formation and release of empty tRNA
what has to happen for translation to be initiated?
small subunit scans mRNA for AUG codon and binds to tRNA-met then recruits the large subunit
what causes translation to end?
release factor binding to A site
what factors control protein concentration within the cell?
rate of translation, rate of degradation by the proteasome
how does the proteasome recognise the trash?
addition of multiple ubiquitin molecules by E2/E3 ubiquitin ligases
what is glycosylation? where in the cell does this protein come from? what is the relevance of this modification?
addition of 14-sugar oligosaccharides.
the ER (possible the golgi, where it is further processed).
it is used to identify whether a protein is folded properly
protein synthesis map
protein synthesis is a series of processes which include transcription which occurs in the nucleus where polymerase uses a DNA sequence (same as gene) to produce a mRNA sequence.
Used in translation by ribosomes to assemble amino acids into a polypeptide which undergoes processing in the golgi apparatus to form proteins
e.g. hormones, membrane proteins, connective fibres, enzymes
facts about ribosomes
they are formed of two subunits
they translate mRNA into protein
they are attached to the endoplasmic reticulum
they are free in the cytoplasm
they are made of rRNA and protein
[1] is [2] into [3] which is then [4] into [5]
DNA is transcribed into mRNA which is then translated into proteins
RNA is ribonucleic acid, but what do the prefix letters stand for?
m – messenger
r – ribosomal
t – transfer
the nucleolus is where…
ribosomal RNA is made
The genes coding for the majority of the ribosomal RNAs are found
clustered on specific chromosomes
where are the ribosomal proteins transcribed
nucleus (by RNA polymerase II) and is exported to the cytoplasm for translation.
How does the cell manage to produce enough rRNA for its needs?
Multiple copies of the rRNA encoding genes expressed at high levels
where are the complete ribosomes (both subunits assembled?
cytoplasm (only when they encounter mRNA)
where are ribosomal proteins translated?
cytoplasm
where are the ribosomal subunits assembled?
nucleolus
Which subunit of the ribosome contains the mRNA binding groove?
small
With which part of the ribosome does the incoming amino acyl-tRNA first interact?
A-site (the E-site is where the empty tRNA resides before leaving the ribosome; the P-site is where the peptidyl-tRNA (i.e. tRNA and attached growing peptide chain) is found)
In what direction is mRNA read during translation?
5’ to 3’
In what direction are proteins synthesised?
Proteins are synthesis from the amino (N) terminus to the carboxy (C) terminus
what is the start codon and what does it encode
AUG and encodes the amino acid methionine ( met or M)
how many stop codons does the standard eukaryotic genetic code contain with example.
three e.g. UAA, UGA, UAG
what is a polyribosome
many ribosomes attached to one mRNA molecule
How do polyribosomes increase the efficiency of translation?
allowing multiple ribosomes to translate a single mRNA molecule at the same time. This process allows for the rapid production of multiple copies of the same protein
the endoplasmic reticulum is composed of…
a phospholipid bilayer/membrane surrounding a space called the lumen . It is contiguous with the nuclear membrane
Name two types of protein which are sythesised into the endoplasmic reticulum.
ER, /secretory/, /transmembranous/, Endoplasmic Reticulum, /Golgi/, /lysosomal/, secreted, transmembrane, lysosome
What is the name of the complex which recognises the ER signal peptide?
signal recognition particle SRP
describe the translocation of a secretory protein into the ER
The SRP binds to the signal peptide and pauses translation/, /stops translation/, /halts translation/, /translation stops/,
Then the SRP attached to the protein which is being synthesised interacts with the SRP receptor and brings the ribosome to the /protein translocator/, /pore/, /translocator/.
A plug blocking the protein translocator/ translocator/pore is displaced allowing translation to proceed directly into the ER lumen/ endoplasmic reticulum. The Signal Recognition Particle and SRP Receptor are displaced, recycled, released.
Amino acids in the signal peptide that sends a protein into the endoplasmic reticulum are typically:
hydrophobic
Hydrophobic amino acids hate water and tend to be buried in the middle of the protein. They also tend to stick in membranes (phospholipid bilayers have a nice charge free, water free environment in the centre). By contrast hydrophilic amino acids love water
True or false: Co-translational translocation into the ER lumen requires no more energy than translation in the cytoplasm?
true
Once the ribosome has docked with the protein translocator the only path open is through the pore and into the ER lumen. As the protein is synthesised and pushed out of the ribosome passes into the lumen without requiring any extra energy.
Name the enzyme which removes the signal peptide from a soluble secretory protein. why is it necessary
signal peptidase, The hydrophobic signal peptide is retained within the membrane
The hydrophobic amino acids found in the signal peptide make it get stuck in the membrane. If a soluble secretory protein is being synthesised, the signal peptide needs to be removed by signal peptidase to release it from the membrane
where does the majority of new membrane synthesis occur
ER
Where are the enzymes which synthesise testosterone mainly housed?
smooth er
Name two cell types in which the smooth endoplasmic reticulum is widespread
muscle, /testes/, /Leydig/, /hepatocyte/, liver, /hepatocytes
sarcoplasmic reticulum
In the muscle cells the smooth endoplasmic reticulum is expanded and has a special name: the sarcoplasmic reticulum. Its role is to store calcium ions which are released through a channel that opens when the action potential from a nerve depolarises the plasma membrane. The rise in levels of calcium ions in the cytoplasm tells the muscle to contract. Once the membrane potential has been restored and the channel closed, abundant pumps in the membrane rapidly return these ions to the SR.
