D1.2: Protein Synthesis Flashcards
How are proteins synthesized?
RNA synthesis occurs in 2 stages:
- Transcription:
DNA is transcribed (copied) and a mRNA molecule is produce - Translation:
mRNA is translated and an AA sequences is produced
What is mRNA?
Messenger RNA
Single stranded RNA molecule that transfers information in DNA from nucleus -> cytoplasm
Carries a message to another part of the cell -> used instead of DNA because DNA too big to fit through nuclear pore
mRNA production requires RNA polymerase
What are the sense and antisense strands?
What are the coding and template strands?
Sense strand:
The strand which the mRNA sequences is a copy of
Coding strand:
The strand of DNA that carries the genetic code
Antisense strand:
The strand which the mRNA is being built on
Template strand:
To get the RNA transcript of the coding strand, the template strand is transcribed to form mRNA
How does DNAs stability contribute to its use as a template?
DNA -> very stable
- hydrogen bonding between bases
- phosphodiester bonds between nucleotides (backbone)
= genetic code is not prone to breaking or changing
Allow single DNA strands to act as reliable templates for transcription
- certain somatic cells -> don’t divide during lifetime -> genetic sequence conserved due to stability
How does transcription relate to gene expression?
Approximately 20k protein-coding genes in human genome
-> but not all are needed (ex: insulin in heart)
= specialized cells can switch certain genes off/on to match requirements of cell -> GENE EXPRESSION
- only genes that are switched on can go through transcription and translation
Many ways to control gene expression
- but transcription = step 1 -> key stage where gene expression can be on/off
What is translation (in the context of protein synthesis)?
Cytoplasm of cell -> ribosomes
Always occur after transcription (needs mRNA)
Involves taking the genetic code from mRNA and synthesizing a polypeptide (sequences of AA that are covalently bonded)
- translate mRNA -> AA
Order of polypeptides based on info stored in the genetic code of mRNA
What is the role of ribosomes in protein synthesis/translation?
Ribosome 2 halves -> large and small
- made of proteins + rRNA (ribosomal)
- assemble together like sandwich on mRNA strand
Small subunit:
Binds to mRNA
Large subunit:
Binding site for tRNA (2 at a time)
Catalyzes peptide bonds between AA
What is the role of tRNA in protein synthesis/translation?
tRNA -> transfer RNA
- free in cytoplasm
- 20 types
- shortest RNA: 80 nucleotides
Translation -> depends on complementary base pairing between codons on mRNA and anticodons on tRNA
tRNA binds with specific AA (that are in the cytoplasm) and brings them to mRNA molecule on the ribosome
Three bases (anticodon) on each tRNA pairs with complementary triplet (codon) on mRNA
Clover structure:
Top -> where AA binds
Left -> sugar-phosphate backbone
Bottom -> anticodons
-> folded structure from H bonds between bases
What is the start triplet/codon?
AA: methionine
Triplet (DNA): TAC
Codon (mRNA): AUG
Start+stop signals: allow cell to read DNA correctly and produce correct sequences of AA
-> genetic code is non-overlapping: each base is only read once
What does the degenerate nature of genetic code mean?
4 bases -> 64 different codons/triplets possible
BUT there are only 20 unique AA
-> code is degenerate: multiple codons/triplets code for the same AA
More than one sequence for AA -> limit the effect of mutations
Explain the movement of the ribosome along the mRNA molecule
in cytoplasm: mRNA attaches to a ribosome
tRNA complementary anticodon forms H-bonds with codon on mRNA
- first one is always methionine
x2
Peptide bond forms via condensation reaction between the 2 AA
- anabolic: requires energy (ATP)
Ribosome moves along mRNA one codon at a time (5’ -> 3’)
During translation: 2 tRNA (with AA) on ribosome at any one time
- Third tRNA bonds -> first tRNA without AA is released
Continues until a stop codon on mRNA reached -> signal for translation to stop
AA chain released to form final polypeptide
What is a gene mutation?
A change in the sequences of bases in a DNA molecule
-> could result in a new allele
-> copying errors that take place during DNA replication
DNA -> determines sequence of AA -> mutations in gene can lead to changes in the polypeptide
Occurs all the time and randomly
Usually harmful/neutral, can be beneficial
How are mutations inherited?
Mutations in normal body cells -> not inherited
Eliminated once the affected cell is dead
Mutations in sex cell/gametes -> inherited by offspring
Lead to heritable genetic conditions
Gene mutation example: sickle cell mutation
In haemoglobin gene:
Mutation in DNA -> GAG to GTG on the coding strand
= mRNA codon is GUG instead of GAG
= during translation AA is valine (val) instead of glutamic acid (glu)
-> results in a change in the 6th position of polypeptide
-> hydrophilic -> hydrophobic AA = extreme change in structure
Global distribution:
Most common in areas with increased malaria
Sub-Saharan africa (80%)
Effects:
HbA allele -> HbS allele = banana RBCs
- Limited O2 carrying capacity
- Block capillaries -> limit blood flow
People with sickle cell anemia/disease ( 2 copies of allele): acute pain, fatigue, anemia
Sickle cell trait (1 copy of allele): relative resistance to malaria
What are the stages of mRNA synthesis (transcription)?
- Initiation:
Short promoter sequence is located before the coding sequence of the gene -> binding site for RNA polymerase
- RNA polymerase causes DNA strands to separate - Elongation:
RNA polymerase moves along the template strand (build in 5’ to 3’)
- RNA polymerase continue to transcribe until terminator sequence - Termination:
RNA polymerase reaches terminator sequence
- trigger detachment of RNA polymerase and mRNA strand
Explain the regulation of gene expression in eukaryotes
Genes -> not expressed equally
- essential genes are expressed all the time (ex for main enzymes in respiratory pathways)
- other one expressed as needed
Regulatory mechanism to ensure correct gene at correct time
Eukaryotes regulate gene expression in response to environment:
Specific proteins bind to DNA to regulate transcription
- ensure only genes required are being expressed at right time, place and amount
General transcription factors: type of transcription factor that binds directly to the promoter to help initiate transcription
- help RNA polymerase attach
- in eukaryotes: many general transcription factors needed
Explain the function of the promoter in transcription
Only certain DNA sequences code for polypeptide -> coding sequences/genes
Non-coding sequences produce functional RNA molecules like tRNA that are involved in the regulation of gene expression
- enhancers, silencers, promoters
Promoter: noncoding sequence located near a gene -> not transcribed
- act as binding site for RNA polymerase during initiation
- controlled by regulatory proteins
What are exons and introns?
Coding sequence/exons:
The regions of the DNA molecule which code for the production of polypeptides
1.5% of human genome
Intron:
Non-coding sequences of DNA found within genes of eukaryotic organisms (diff proteins can be made depending on how introns removed)
Majority of eukaryotic genome -> non-coding regions
- have regulatory sequences that control gene expression by enhancing/suppressing transcription
- make tRNA, rRNA
What are telomeres?
Regions of repeated nucleotide sequences at the end of chromosome that provide protection during cell division
Facilitated binding of RNA primer at the end of the chromosome
Without telomeres: DNA replication cannot continue to end of DNA -> chromosome shorter each cell division
Telomeres shorten with age due to oxidative damage within cells
- loss of telomeres can be caused by:
Smoking, exposure to pollution, obesity, stress, poor diet
- loss can be reduced by:
Antioxidants in diet
Why do eukaryotic cells need post-transcriptional modification of mRNA?
Post-transcriptional modification of mRNA:
Prevent degradation (mRNA inherently unstable)
Increase efficiency of protein synthesis
Expands complexity of the proteome
Transcription and translation occur in separate areas -> mRNA has time to degrade
Why do prokaryotic cells not need post-transcriptional modification?
In prokaryotes transcription and translation can occur immediately after one another -> prevents degradation of mRNA
What are the three steps of post-transcriptional modification in eukaryotic cells?
- Methylated cap added to 5’ end to protect against degradation by exonucleases
- A poly-A tail (long chain of adenine) added to 3’ end for protection and to help mRNA exit nucleus
- Non-coding sequences (introns) are removed and coding sequences (exons) are joined together
What does splicing do?
Transcription: whole gene transcribed (introns and exons)
- but introns not translated -> don’t code
So before pre-mRNA exits nucleus SPLICING occurs:
- introns removed
- exons joined together
- carried out by splicing enzymes (spliceosome)
Result: mature mRNA that contains only exons
What is alternative splicing?
The exons in general can be spliced many different ways -> produce many different mature mRNA molecules from same gene through alternative splicing
Certain exons not incorporated into final mature mRNA
-> Polypeptide translated from alternatively spliced mRNA -> different AA sequence -> different protein with different structure + function
Reason why proteome > genome
What are the different binding sites of the ribosome?
Large subunit -> 3 biding sites (E, P, A)
A (aminoacyl):
mRNA codon joins with the tRNA anticodon
P (peptdiyl):
AAs that are attached to tRNA joined by peptide bonds
E (exit):
tRNA exits the ribosome
Explain the initiation step of translation
Preinitiation:
1. An activating enzyme binds tRNA with the anticodon (for start UAC)
- Activating enzyme attaches AA to the tRNA (for start methionine -> initiator tRNA)
- tRNA can now bind to codon (initiator tRNA -> start codon -> begin translation)
Initiation:
1. Small subunit binds to 5’ end of mRNA
- Initiator tRNA (methionine) binds to P site of small subunit
- Ribosome moves along the mRNA (5’ -> 3’) until it locates a start codon (AUG)
- Large subunit of the ribosome binds to tRNA and small subunit
Explain the elongation and termination steps of translation
- A second tRNA (with AA) that is complementary to second codon on mRNA binds with A site of ribosome
- The AA on the tRNA in the P-site (first tRNA) -> transfer AA to tRNA in A-site (second tRNA)
- done via formation of peptide bond (condensation) - Ribosome moves 1 codon along mRNA (5’ -> 3’)
- tRNA in P-site (first tRNA) moves to E-site -> released
- tRNA in A-site (second tRNA) moves to P-site - Next mRNA codon exposed -> tRNA complementary to next codon in mRNA -> binds to A-site
- AA becomes part of polypeptide chain - Steps 6-8 repeated until stop codon
- When stop codon -> translation stop:
- release factor attach to A-site instead of tRNA
- polypeptide chain released
- ribosome complex disassemble (reused)
What are examples of how proteins can be modified immediately after translation?
Protein folding: secondary, tertiary, quaternary structures
- disulfide bonds
- molecular chaperones (prevent incorrect folding)
Conjugation with other protein or inorganic cofactors: quaternary structure
- haeme in haemoglobin
Chemical modification: improve stability or activity
- glycosylation
- phosphorylation
Removal of AAs from polypeptide chain: proteolytic cleavage
Insulin as an example of post-translation modification
When insulin first synthesized -> pro-preinsulin (110 long polypeptide chain), attached to walls of ER
Modified by enzyme in RER -> remove signal peptide from end -> detach from ER -> proinsulin
Proinsulin to Golgi apparatus -> Proinsulin fold + disulfide bonds formed -> cleaved (C peptide (linking sequence of AAs) removed from middle) -> 2 chains (A-chain and B-chain) formed (attached by disulfide bonds) -> MATURE INSULIN
Insulin packaged into vesicles -> stored and used as needed
What is a proteasome?
Unneeded, damaged, misfolded proteins -> recycled in the body -> necessary to sustaining functional proteome
- involves PROTEASE ENZYMES to break peptide bonds in proteins -> release AA to be used in translation -> new proteins
- called proteolysis
Proteasome is organelle in eukaryotic cells where this occurs
- containing protease in organelle -> prevent break down of working proteins
Proteins that need to be recycled tagged by chemical UBIQUITIN -> starts process of them being broken down in the proteasome
What is the central dogma?
The idea that one gene corresponds to one polypeptide chain and therefore one protein
Why is the central dogma not true?
- Most of the genome isn’t genes:
Genes -> section of DNA used to synthesize polypeptides
Most of genome -> introns (control gene expression, telomeres) - Once a gene is transcribed, mRNA can be modified
- alternative splicing -> many proteins from same mRNA
