Cell Biology Chapter 8 - Protein Synthesis and Sorting Flashcards
The Central Dogma of Molecular Biology
DNA polymers code RNA polymers which code Amino Acid polymers
Three challenges
- DNA to RNA or RNA to protein subject to mistakes?
- How do cells ensure the right information occurs at the right time and place?
- How does a cell ensure information is efficiently passed from one form to another?
Transcription
Converts the DNA Genetic Code into RNA
The major difference between DNA replication and Transcription
Transcription is HIGHLY selective
*Only defined portions of DNA are transcribed to RNA
RNA Polymerases
Transcribe Genes In A “Bubble” Of Single-stranded DNA
Transcription bubble
Similar to replication bubble
- Unidirectional
- Single strand
*On rare occasions in prokaryotes, two replication bubbles can cross in opposing directions to read different templates on different DNA strands
Transcription Occurs In Three Stages
- RNA is synthesized 5’3’ and the DNA is “read” in the opposite direction
- In eukaryotes, three different RNA polymerases are used to transcribe different forms of RNA
RNA polymerase I, II, II (pol I, II, III)
Pol II makes mRNA and is the most studied
Transcription Begins After
A RNA Polymerase Binds To A Promoter Site On DNA
- Prokaryotic RNA polymerase is simple
- Eukaryotic RNA polymerase has about 12 subunits
Location of Promoter sites
Prokaryotic promoters are short and lie close to the actual transcription start site
Eukaryotic promoters may be spread out over hundreds of DNA base pairs
Transcription Factors
Can bind DNA or other DNA-binding proteins
Basal Transcription factors or the Basal Transcription Complex are the smallest group of proteins required to produce any transcription
6 factors make up the basal transcription factors
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH
- Some of these are made of multiple polypeptide subunits each
Transcription: Initiation
RNA synthesis begins when basal transcription factors bind to the promoter nearest to where transcription begins
*This is NOT mark the beginning of translation
Core promoter
(essential for transcription) on which the basal transcription factors assemble
Assembly of the Initiation Complex (continued)
TFIID is recruited to the TATA box
TFIIH
- binds specifically the template strand and it also has helicase activity
- With TFIIE it “caps” RNA Pol II and holds it in place
- It has kinase activity and phosphorylates RNA Pol II activating it
Order of TF adding sequence
- TFIID (has affinity for the minor groove of the DNA) —>
- TFIIA
- TFIIB
- TFIIF
- TFIIE
- TFIIH
RNA polymerase only appears after all the basal transcription factors have been assembled.
TFIIH
The “H” stands for helicase activity and helps unwind it. Phosphorylates RNA pol and activates it.
TFIIE
“E” Binds hold RNA poly II and holds it in place. “The Escape claw”
RNA transcript
transcript is extended in the 5’-to-3’ direction as the RNA polymerase reads the template DNA strand in the 3’-to-5’ direction
The entire replication bubble is within the space occupied by RNA pol II
Supercoiling Solved by Two Enzymes
Gyrase reverses extra positive supercoiling ahead of the transcription bubble by inducing negative supercoiling
Topoisomerase relaxes negative supercoiling behind the transcription bubble
Stage 3 of Transcription: Termination
Termination results in the release of the mRNA transcript and RNA pol from the DNA template
Prokaryotes and Eukaryotes use different termination methods
Three features of termination are especially important
Three Important Features of Termination
- Prokaryotes (not eukaryotes) have termination encoded within the DNA sequence : called terminators
Fold into hairpin loops of semi-stable double-stranded RNA
2) Bacterial rho protein is an example of an RNA pol binding-protein necessary for termination of some transcription
3) Terminators are not universally effective
Anti-terminators can suppress termination
Polycistronic RNA and operons result when adjacent or clusters of genes are transcribed on a single RNA molecule
One of the most striking differences between Prokaryotic and Eukaryotic transcription is that mRNA structure is modified by Eukaryotes prior to their use
True
The Spliceosome Controls RNA Splicing
Introns are removed from the primary transcript and exons are stitched together in a transesterification reaction at the exon-exon boundary
Alternative splicing can generate a large variety of proteins from a single DNA sequence
RNA Modifications
- 5’ methylguanosine cap
- Poly(A) tail
Eukaryotic mRNA has a methylated 5’ cap
The cap protects the 5’ end of mRNA from nucleases and may be methylated at several positions
RNA Export
- poly(A)-binding protein (PABP) at 3’ end
- Sometimes the mRNA with all associated proteins is called the heterogeneous nuclear ribonucleoprotein particle (hnRNP)
- After RNA processing is complete, also called the messenger ribonucleoprotein particle (mRNP)
Proteins Are Synthesized By ______?
Ribosomes Using An mRNA Template
Translation
is the term used to describe the conversion of mRNA information into polypeptides
Translation requires cooperation
between ribosomal RNAs, transfer RNAs, messenger RNAs, and numerous proteins.
The steps of translation are grouped into three stages:
initiation, elongation, and termination
The Stoke’s Radius
Radius is Based on the Time it Takes to Migrate Through a Gel
Prokaryotic Ribosomes Typically 70S (composed of 50S and 30S subunits)
Eukaryotic Ribosomes Typically 80S (composed of 60S and 40 S subunits)
Aminoacyl tRNAs
Contain a three-letter “anti-codon” that will match up with the three-letter “codon” on the mRNA
Aminoacyl tRNA synthetase uses ATP energy to couple the amino acid to the hydroxyl group of the 3’ end of the tRNA
This “charges” the tRNA with the energy needed to generate the peptide bond!
Translation Factors
- Initiation Factors and Elongation Factors
- Form a critical link between RNA processing and translation
- Found in eukaryotes and prokaryotes
The Ribosome Has Three tRNA-binding Sites
A Site – is where the aminoacyl tRNA first attaches to the ribosome
P Site – is where the newly arrived amino acid is removed from its charged tRNA and added to the growing chain
E Site – Is where the tRNA is ejected from the ribosome
Stage 1of translation: Initiation requires base pairing between mRNA and rRNA
Goal = bring all of the elements necessary for translation together into a giant cluster
Once the mRNA and small subunit are properly aligned, the first tRNA (initiator tRNA) binds to the AUG, and the large ribosomal subunit clamps down on the small subunit, forming an intact ribosome
Prokaryotic Initiation
Bacteria use a specific mRNA sequence called the Shine-Delgarno sequence that base pairs with a portion of the rRNA on the small subunit
Shine-Delgarno sequence is upstream of a translation start sequence called the initiation site
-5’-AUG-3’
Eukaryotic Initiation
5’-methylated G cap on mRNA binds eIFs (eukaryotic initiation factors)
Once the cap is identified, the small ribosomal subunit crawls towards the initiation site in the 3’ direction
Since AUG is a common sequence, it doesn’t necessarily stop at the first one it sees. Surrounding sequence helps it.
At least a dozen different translation factors attach to the complex at this time.
Stage 2: ElongationProkaryotic Elongation as a Model
Proofreading Step
EF-Tu
Ensures a perfect codon/anticodon match
Peptide Bond Formation
Peptidyl-transferase is part of the large ribosomal subunit
Moving On
EF-G-GTP provides the energy to shove the ribosome forward a codon
Chain Elongation
Occurs at the P site
Translation stop sequences
UAA, UAG, UGA
Do not bind to any tRNAs
Instead, these bind release factors that fit into the A-site on the ribosome
occurs when the bond holding the polypeptide to tRNA is hydrolyzed
Water is the acceptor in this hydrolysis reaction
Three Important Themes
The sequence of bases in the coding region of a gene is directly reflected by the sequence of amino acids in a protein
Differences in amino acid sequence result in great variation in protein shape
Controlling protein shape is the mechanism cells use for controlling protein location
Controlling protein shape is the mechanism cells use for controlling protein function
Signal Sequences Code For Proper Targeting Of Proteins
Important: The Cytosol is the default destination for all proteins synthesized there
Proteins without signal sequences stay in the cytosol
Proteins with signal sequences are transported
“ticket analogy”
Nuclear Transport
Dilemma: How to keep Chromosomes in and Organelles out?
Transcription enzymes need to get in and mRNA needs to get out
Consider the following statements:
i. The origin of replication is the site where transcription of a gene begins.
ii. The basal transcription complex binds to the promoter of a gene.
iii. RNA polymerase is modified by phosphorylation, DNA polymerase is not.
iv. The DNA replication fork contains two DNA polymerase enzymes.
v. The pre-initiation complex on DNA establishes whether a gene will be transcribed or not.
Which of these are true?
ii, iii, and iv
Central Dogma of Molecular Biology
DNA —> RNA —> Amino Acids —> Proteins
DNA transcription is always in the ______ direction
3’ -to- 5’
There are no primers
Transcription stages
Initiation —> Elongation —> Termination
Eukaryotes express three different RNA polymerase. Each specialized to generate a different kind of RNA molecule
RNA pol I
RNA pol II
RNA pol III
RNA pol I
synthesizes a single large ribosoma RNA which is later cut into the 3 parts of the rRNA found in ribosomes.
RNA pol II
synthesizes messenger RNAs
RNA pol III
synthesizes tRNAs
Promoters
Mark the designated sites of DNA transciption
NB// Where as replication begins at sequences called “origins of replication”, transcription begins at sequences called promoters.
Proteins that control transciption are called ________
transcription factors
Basal transciption factors
they literally form the base upon which the regulatory structures are built.
For RNA pol II, six factors make up this complex
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH
Transcription: Initiation
begins when basal transcription factors bind to the promoter nearest to where transcription begins. This site is called the transcription startpoint
The promoter is subdivided into distinct sequences. The portion near the transcription start point is called _______
core promoter because its is absolutely essential for transciption
The core promoter is called
TATA box.
5’ -TATAA- ‘3
The TATA box binds TFIIID
Basal transciption factors binding sequence
TFIIID + 3 other BTF
These form a pocket for RNA pol II
After pol II binds TFIIIE & TFIIIH attach to cap the polymerase.
TFIIIH —>
Ensure pol II reads the right template.
Contains kinase enzyme that add phosphate and initiate conformational change. BTF are the activated
TFIIIE —>
partially unwinds DNA double helix
RNA pol has no proof reading abilities
True
Stage 2 of Transcription: Elongation
RNA transcript is extended in the 5’-to-3’ direction as the RNA polymerase reads the template DNA strand in the 3’-to-5’ direction
Supercoiling Solved by Two Enzymes
Gyrase reverses extra positive supercoiling ahead of the transcription bubble by inducing negative supercoiling
Topoisomerase relaxes negative supercoiling behind the transcription bubble
Stage 3 of Transcription: Termination
Prokaryotes and Eukaryotes use different termination methods
I prokaryotes its encoded by specific sequences called terminators.this forms a sei stable double-stranded RNA called hairpin loop.
Termination - may require additional protein
some terminators require additional proteins (Rho protein in bacteria) to bind RNA polymerase to induce shape change and stop transcription.
Terminators are not universally effective
Terminators are not universally effective
Anti-terminators can suppress termination
Polycistronic RNA and operons result when adjacent or clusters of genes are transcribed on a single RNA molecule
RNA processing
One of the most striking differences between Prokaryotic and Eukaryotic transcription is that mRNA structure is modified by Eukaryotes prior to their use.
The Spliceosome Controls RNA Splicing
RNA Splicing
Nucleotide sequences at the boundries of of introns and exons on mRNA facilitae the breakng and forming of the suger phosphate backbone by a process called transeterification
Alternative splicing
Alternative splicing can generate a large variety of proteins from a single DNA sequence by “mixing & matching” exons after introns have been spliced out.
RNA Modifications
5’ methylguanosine cap
poly(A) tail
RNA Export
Is Unidirectional And Mediated By Nuclear Transport Proteins
RNA Export: Function of the Poly A-tail
poly(A)-binding protein (PABP) at 3’ end
mRNA with all associated proteins is called
…..the heterogeneous nuclear ribonucleoprotein particle (hnRNP) or ribonucleoprotein particle (mRNP)
Translation Occurs In Three Stages
initiation, elongation, and termination
The first goal of initiation is for the ribosomal subunit to find the ______
Ribosomal binding site (Specific translation start point)
Bacteria and eukaryotes accomplish this differently
Ribosomal binding site in bacteria is the _____
Shine Delgarno sequence…..forms complementary pairing with a section of the small subunit of the ribosome.
5’ -AUG- 3
Once small subunit of ribosome pairs with shine Delgarno sequence _______
1st tRNA called initiator tRNA binds to the AUG
Prokaryotic Ribosomes size _____
Typically 70S (composed of 50S and 30S subunits)
*based on The Stoke’s Radius
Eukaryotic Ribosomes size _____
Typically 80S (composed of 60S and 40 S subunits)
Eukaryotes dont have a Shine Delgarno sequence.
True. instead they capitalize on the 5 methylguanine cap that is added to the 5’ of the mRNA.
Small subunits in eukaryotes bind to initiation proteins (eIFs) on the 5’ methylguanine cap
elongation - Three inputs are required
- Charged tRNA linked to an amino acid
- EF-Tu-GTP protein
- Translation factor complex EF-G-GTP
Stage 3: Termination
Translation stop sequences: UAA, UAG, UGA
Do not bind to any tRNAs
Instead, these bind release factors that fit into the A-site on the ribosome
occurs when the bond holding the polypeptide to tRNA is hydrolyzed
Water is the acceptor in this hydrolysis reaction
What cellular activity does the term gene splicing describe?
It describes the removal of nucleotides from a primary transcript RNA
What distinguishes transport of proteins into the nucleus from other modes of protein transport?
Proteins targeted to the nucleus are transported in a fully folded, functional state
Which statement best explains the difference between transcription and translation in eukaryotic cells?
Transcription occurs in the nucleus, translation occurs in the cytosol.
Removal of a TATA box from a eukaryotic gene would likely result in which of the following outcomes?
No transcription of that gene
Which statement best explains the difference between transcription and translation?
Transcription copies one nucleotide sequence to another, translation converts a nucleic acid sequence into an amino acid sequence.
During the elongation phase of translation, what occurs at the P site on a ribosome?
A peptide bond is formed between adjacent amino acids in the growing polypeptide
Which statement most accurately describes how translation is terminated?
Release factor binds to the stop codon, so the P site cannot form any more peptide bonds.
Signal Sequences Code For Proper Targeting Of Proteins
The Cytosol is the default destination for all proteins synthesized there.
Proteins without signal sequences stay in the cytosol
Proteins with signal sequences are transported
“ticket analogy”
Nuclear Transport
Dilemma: How to keep Chromosomes in and Organelles out?
Transcription enzymes need to get in and mRNA needs to get out
signal sequences that permit entry to and exit from the nucleus are called ______
Nuclear localization sequences (NLS) nad nuclear export sequences (NES). These bind to receptor proteins Importin and Exportins
Nuclear Transport is Bidirectional
Proteins transported in/out of nucleus in folded, functional state
Nuclear Transport
Simple Diffusion
Colloidal Gold diffusion into nucleus was inversely related to particle diameter (10 nm or larger were entirely excluded)
Small particles diffuse in through tiny, 8 aqueous diffusion channels in the pore complex
Thought to be permeable to ions and small proteins and ATP, GTP, CTP, TTP used for DNA replication
Nuclear Import – Step by Step
- Importin recognizes proteins with an NLS sequence
This Importin-cargo complex goes through the nuclear pore complex - Ran-GTP (a small G protein) binds to Importin causing release of the cargo
Ran-GTP bring importin back to the nucleus for another round
GTP hydrolysis triggers release of importin back in the cytosol
Nuclear Export – Step by Step
Usually RNA, rather than protein
Proteins with nuclear export signals (NES) bind RNA
Exportin binds Ran-GTP
This triggers cargo loading onto Exportin
Cargo-Exportin-Ran-GTP travels through the nuclear pore complex to the cytosol
Complex falls apart with GTP hydrolysis
Exportin goes back to the nucleus for another round
Proteins Targeted To The Peroxisome contain ________
Contain Peroxisomal Targeting Signals (PTS)
Peroxisome functions include
- Oxidation of long-chain fatty acids
- amino acids synthesis and breakdown,
- Glycerol synthesis and
- degradation of dangerous molecules called reactive oxygen species (ROS)
Aminoacyl tRNA synthetase:
forms a covalent bond between amino acids and tRNAs in the cytosol
Protein import into the peroxisome most closely resembles protein import in:
the chloroplast
If a cell was transfected with a plasmid encoding Green Fluorescent Protein, and the result was accumulation of GFP in the nucleus, which statement best explains this result?
The GFP gene was engineered to include a Nuclear Localization Sequence.
Consider the following statements:
i. The origin of replication is the site where transcription of a gene begins.
ii. The basal transcription complex binds to the promoter of a gene.
iii. RNA polymerase is modified by phosphorylation, DNA polymerase is not.
iv. The DNA replication fork contains two DNA polymerase enzymes.
v. The pre-initiation complex on DNA establishes whether a gene will be transcribed or not.
Which of these are true?
ii, iii, and iv
What is the function of aminoacyl-tRNA synthetase?
It adds an amino acid to tRNA.
Which statement best describes the peptidyl transferase function of a ribosome?
It adds the growing polypeptide to the aminoacyl tRNA
Which statement best describes the core promoter?
It encodes the sequences that promote expression of the core proteins in the nuclear pore complex
Transport into the ER
Secreted Proteins And Proteins Targeted To The Endomembrane System Contain An ER Signal Sequence
Transport into the ER
Transport into the ER is cotranslational (as it is being synthesized by the ribosome)
Transport into the ER requires that the protein be in its unfolded state
Transport into the ER is unidirectional
As Proteins Enter the ER Lumen, They May Be Post-Translationally Modified
Terminally Misfolded Proteins In The ER Are Degraded In The Cytosol
Misfolded polypeptides in ER are “reverse translocated” back into cytosol
Once in the cytosol, misfolded polypeptides are ubiquitinated and subsequently degraded by proteosomes
Process of identifying, reverse translocating, and destroying these polypeptides is called ER-assisted degradation (ERAD)
The Unfolded Protein Response (UPR) is triggered when unfolded proteins begin to accumulate in the ER lumen
Heat shock proteins to the rescue!
NLS
Nucleus localization sequence
NES
Nucleus Export Signals
RAN
Controls the direction nucleus transport
Proteins Targeted To The Peroxisome
Peroxisome Contain Peroxisomal Targeting Signals (PTS)
True / False
Proteins are transported into the peroxisomal matrix in their properly folded, functional state
True
ERoS
ER Signal Sequence
Secreted Proteins And Proteins Targeted To The Endomembrane System Contain An ER Signal Sequence
True
True or False
Transport into the ER requires that the protein be in its unfolded state
True
Transport into the ER is unidirectional
Transmembrane Proteins
There are The four classes of transmembrane proteins, according to the Singer classification system.
C N N (or C) N (or C) / C (or N)
| | |
N C C (or N)
SRP
Signal recognition particle (Contains 6 subunits and forms RNA called small cytosolic RNA. Uses GTP
Proteins destined for any portion of the endo membrane system express at least one signal sequence called _________
ER signal sequence
discuss the process from SRP —–> translocation.
Go…
As Proteins Enter the ER Lumen, They May Be Post-Translationally Modified
N-linked glycosylation transferase in the translocon attaches clusters of suger called core oligosaccharides to some polypeptides.
Then…
Chaperone Proteins Assist in the Proper Folding of ER Proteins. BiP (Binding proteins) binds to exposed hydrophobic patches in recently translocated proteins.
reverse translocated
Misfolded polypeptides in ER are “reverse translocated” back into cytosol
Once in the cytosol, misfolded polypeptides are ubiquitinated and subsequently degraded by proteosomes
ER-assisted degradation (ERAD)
Process of identifying, reverse translocating, and destroying these polypeptides is called ER-assisted degradation (ERAD)
Unfolded Protein Response (UPR)
The Unfolded Protein Response (UPR) is triggered when unfolded proteins begin to accumulate in the ER lumen
Heat shock proteins to the rescue!
Cytosolic Proteins Targeted To Mitochondria Or Chloroplasts Contain An _________
N-terminal Signal Sequence.
Signal Sequences
Mitochondrial signal sequences
Chloroplast transit peptides
Bind Membrane Protein Receptors (TOMs and TIMs)
Transport is post-translational and in an unfolded state
Requires ATP energy
The Cytoskeleton Immobilizes And Transports mRNAs
mRNAs were previously thought to float through the cytosol
mRNAs preferetially localize to certain areas of the cytosol
They associate with actin filaments and microtubules and are transported by motor proteins
Example – zipcode sequence in b-actin mRNA
