Cell bio exam #3: Translation and Translational Regulation

Codons

A sequence of 3 nucleotides

(read by ribosomes and tRNA)

Read 5’-3'

Each codes for a specific amino acid

UTR

Untranslated regions

Present at the 5’ and 3’ ends

Proteins are not made from these regions

UAA, UAG, UGA

The 3 stop codons

Open reading frame (ORF)

The region that codes for amino acids 

tRNA’s

Serve as adaptors between the codons and amino acids

Made up of modified bases

Fold up into shapes that allow it to be plugged into the ribosome during translation

Derived from genes

Contain a single stranded 3’ overhang where amino acids are attached

Made up of many modified bases

Made up by RNA polymerase III

Goes through many processing steps to help stabilize and help with structure and folding, making them ready to receive amino acids

Can be recycled and reused

Histidyl tRNA synthetase

A protein that binds the amino acid histidine to the correct tRNA with the correct anti-codon

Transfers a histidine to the tRNA

Binds to the 3’ single-stranded overhang of the tRNA

Part of the aminoacyl tRNA synthetase family

Aminoacyl tRNA synthetase

protein that allows amino acids to attach to tRNA

There is a different enzyme under this family of enzymes for every amino acid

There are 20 different ones under this family 

Each recognizes: 1. a particular amino acid; 2. the “cognate tRNAs” to which the amino acid should be attached

Charged tRNA 

The combination of a tRNA with an amino acid attached to its 3’ overhang tail

Inosine

A modified adenosine base

Is able to base pair with uridine, cytosine, or adenine due to its modifications

created by deaminating adenosine, where the adenine bases amino group is replaced with a keto group, specifically transforming it into hypoxanthine (which is the base that makes up this nucleotide) 

Usually found in the first position of the anticodon on tRNA

Only found in tRNA (NOT in mRNA)

Can bond to more than one type of base —> always bonds with 2 hydrogen bonds

The wobble effect

A phenomenon that occurs when tRNA and mRNA are put together —> the first 2 nucleotides are very tight together —> the third nucleotide of the mRNA and the first of the tRNA are more spread apart (more free to move- can wobble)

Causes the last nucleotide pairing to be less important allowing for weird base pairing because the hydrogen bonding is not as strong —> nucleotides are more free to move

The base in the first position on the tRNA (the 5’ end) is usually an abnormal base like inosine, pseudouridine, tyrosine, etc. —> these abnormal bases can pair with more than one type of base in the third position of the codon of the mRNA

The tRNA is able to wobble relative to the mRNA

Ribosomes

Make peptide bonds

Consist of a large subunit and a small subunit —> both subunits are made up of a mixture of proteins and rRNAs

Build polypeptides one amino acid at a time

Require “help” from proteins to carry out translation via three distinct stages —>proteins are constantly going in and out 

Functionally very similar in both prokaryotes and eukaryotes (structurally a little different) 

Peptide bonds

A type of covalent (strong) bond made between amino acids through a dehydration reaction (losing water)

Join together amino acids

Ribosome small subunit

The part of a ribosome whose primary job is to help find the AUG (start codon)

In prokaryotes, contains a Shine-Dalgarno sequence which allows for the recognition of the AUG

In eukaryotes, the ribosomal subunit starts by recognizing the 5’ methly-cap of the mRNA, then scanning for the AUG (could potentially be thousands of nucleotides between the 5’ methyl cap and the AUG)

Ribosome large subunit

The part of a ribosome that forms the peptide bonds between amino acids

rRNA’s here catalyze peptide bond formation

Made up of an E, P, and A site

Shine-Dalgarno sequence

A complementary nucleotide sequence in prokaryotic mRNA directly upstream from the start codon (AUG)

Guides the small subunit of a ribosome to an AUG

Short sequence of ~7 nucleotides

Initiation (translational) 

Ribosomes bind at the 5’ UTR and initiates polypeptide synthesis at the start codon 

Only requires the small subunit until AUG is found

Requires several “eukaryotic initiation factors” (eIFs)

Elongation (translational)

Polypeptide chain elongates by successively adding amino acids

Continues from the start codon (AUG) to the stop codon 

The building of the polypeptide chain by the ribosome (the large subunit) 

mRNA is pulled through the ribosome using eEFs

Requires several “eukaryotic elongation factors” (eEFs)

Termination (translational)

When a stop codon is encountered

Polypeptide is released and the ribosome dissociates from the mRNA

Requires “release factors”

Allows the polypeptide to then free fold into its functional conformation

eIF2

“eukaryotic initiation factor” that binds the initiator to methionyl-tRNA (every protein starts with a methionine —> majorly important for the initiation of translation)

Plugs the methionyl tRNA into the ribosome —> takes energy from GTP

Regulated by binding to GTP or GDP

When GTP bound, it is active; when GDP bound, it is inactive

Can be regulated in order to stop translation generally —> can be inhibited by phosphorylation when cells are in stressful conditions (ex. absence of growth factors) 

Poly-A binding protein (PABP)

Initiation factor that binds the 3’ poly-A tail to the mRNA

Checks the mRNA to make sure that it is ready for translation

Regulates translation rates

eIF4E

“eukaryotic initiation factor” that specifically binds the 5’ cap to the mRNA

Checks the mRNA to make sure that it is ready for translation

Regulates translation rates

Initiation of translation at internal ribosome entry sites (IRES)

A process in which viral eukaryotic mRNAs can initiate translation independent of the 5’ methyl cap

Requires ATP

A “shortcut” to initiating translation

P (peptidyl) site

A (aminoacyl) site

E (exit) site

The three parts of the large subunit of the ribosome

eEF2

Eukaryotic elongation factor in charge of the process of translocation

A GTP binding protein —> uses GTP to move the mRNA chain through the ribosome

Release factors

Proteins that take on a certain shape (similar to that of tRNA) allowing them to recognize stop codons and stop the process of translation by disrupting the process

eEF1a (alpha)

Eukaryotic elongation factor in charge of plugging each amino acid into the tRNA (except for the first one- methionine)

GTP binding protein —> uses energy from GTP to attach amino acids to tRNA and stuff it into the ribosome

Able to be recycled and reused

Guanine exchange factors (GEF)

A family of proteins that carry out the exchange of GDP for GTP by pushing GDP off of the GTPase and allowing GTP to preferentially bind to the GTPase (because there is a lot of GTP available in cells- in excess)

Promotes the exchange of GDP for GTP to activate GTP-binding proteins

Note: GDP is NOT changed to GTP because of the transferring of phosphates

Also called eEF1By

ONLY expressed in the nucleus

GTPase activating protein (GAP)

Protein that accelerates the hydrolysis of GTP to GDP

GTP-binding turning off proteins 

Opposite of GEFs

Speeds up GTPase (GTP binding protein) activity

ONLY expressed in the cytosol

Anti-codon

Sequences that run 3’-5’

Anti-parallel to the mRNA codon sequences

RNA polymerase I and III

The polymerases responsible for coding for rRNAs

There are 4 subunits to a ribosome —> 3 are made up by I, the other (5s subunit) is made up by III

Aminoacyl AMP

An intermediate created by ATP which modifies the amino acid and gets it ready to be attached to the tRNA

Polypeptide

Formed by the joining of many amino acids by peptide bonds

Always synthesized in the amino —> carboxyl terminus direction

Amino terminus & carboxyl terminus

The 2 distinct ends of an amino acid

Svedberg units

Sedimentation values found from isolating and purifying ribosomes, spinning them down a gradient, and allowing them to settle at different spots in a centrifuge tube

Allows for assessing the size of a ribosome

28s, 18s, 5.8s, and 5s

The 4 different rRNAs that make up the large and small subunit of a ribosome are _________

5s

The svedberg unit making up part of the large ribosomal subunit

Transcribed in the nucleus by RNA polymerase III

Many more copies of this compared to the other svedberg units

Pre-rRNA transcript

Processed via cleavage into mature rRNAS

Gets cut into 3 pieces

Gets recycled and reused for the purpose of helping the start of rRNA synthesis

Preventing it from being degraded and helping with its structure

rRNAs get modified for the main purposes of _________ and __________

Nucleolus

The major site of ribosomal synthesis

Where the different subunits of the ribosome get put together

Does not fully complete the synthesis of a ribosome however —> the rest is done in the cytosol

Mono-cistronic

Word used for describing the organization of eukaryotic mRNA 

—> means that the mRNA is made up of 1 gene, 1 mRNA, 1 protein

Poly-cistronic

Word used to describe the organization of prokaryotic mRNA

—> made up of multiple proteins and translation start sites (ribosomes can start translation from each of the start sites) 

Stop codon

Where the process of translation stops

Kinases

Enzymes that add phosphates generally transiently (temporarily)

Include 2 substrates: amino acids and ATP

Include pockets that allow small molecules to get into them and inhibit certain actions post-translation

There are two different families of these enzymes: serine/threonine and tyrosine

Catalyze phosphorylation at the recognition motif of the protein bound to it

Make really good drug targets due to its pockets

Docking site

A second site on a kinase, separate from the active site,

Includes negatively and positively charged amino acids

Glycogen phosphorylase

An enzyme and substrate that becomes activated when it conformationally changes due to phosphorylation 

A dimer —> made up of 2 different protein subunits 

When inactive, its catalytic site is hidden

When phosphorylated, both sites (one in each dimer) become negatively charged, and become attracted to positively charged amino acids —> this pulls the amino acids towards its catalytic sites, making the enzyme active

Nuclear envelope

Made up of an inner and outer membrane (2 phospholipid bilayers and lumen), the nuclear lamina, and the nuclear pore complex

DNA closer to this part of the nucleus

Perinuclear space

The space in between the two membranes surrounding the nucleus

Continuous with the lumen of the ER

AKA the lumen 

Nuclear lamina 

The only structure within the nucleus that holds its round, sphere-like structure 

Made up of individual proteins called lamins 

Coiled coil dimer

The intertwined lamin polypeptide structure, which then forms head to tail associations 

Hutchinson-Gilford progeria syndrome

An inherited tissue-specific disease caused by mutations in lamin genes

A pre-mature aging syndrome

The basis of the pathologies of this disease and other related nuclear lamina diseases are still unclear

Emerin & LBR

Proteins located on the inner membrane of the nucleus which interact with each other and with the lamina through protein-protein interactions

Mutations in these genes can lead to progeria

Are plugged into the membrane by a lipid anchor (prenylation)

Interact with histone proteins allowing them to interact with chromatin

Mutations in one of these proteins is likely to change the conformation of where the chromatin is in the nucleus, likely changing the accessibility of genes to be transcribed —> leading to a change in phenotype

Nuclear localization signals (NLS)

A short peptide sequence that helps with the identification of molecules that are destined to be transported into the nucleus

Necessary for the transport of materials to the nucleus

This sequence alone is sufficient to take materials to the nucleus

Can be separated into two parts (“bipartite”)

Generally rich in basic amino acids (ex. lysine)

Some examples of these sequences include T antigen, p53, Erg1, and GATA

Acts as a “tag” that can be recognized by a transporter protein (importins) which then import the protein to the nucleus

Eukaryotic initiation factors

A bunch of proteins necessary for starting translation

These proteins work together and form a protein complex that guides the small subunit of the ribosome from the 5’ methyl cap to the AUG (start codon)

Use ATP to unfold mRNA, allowing the small subunit to scan for AUG (*ONLY PART OF TRANSLATION THAT USES ATP —> the rest is GTP)

Allow the tRNA to be “stuffed” into the ribosome

eIF5B

“eukaryotic initiation factor” in charge of assembling the entire ribosome (the small subunit with the large subunit) 

Uses GTP to combine the two subunits of the ribosome together

GTP binding proteins (GTPase)

Proteins that use GTP in order to carry out their functions

GTP acts as an allosteric modulator to these proteins —> change their conformation, determining if the proteins are active or inactive

Always active when bound to GTP and inactive when bound to GDP

Enzymes that break GTP (through hydrolysis) and use the energy created through that to carry out its function

Get help from other proteins to go from GTP —> GDP (active —> inactive)

Each of these proteins have an associated GEF protein that helps with the transition between GDP and GTP

Enzymes that hydrolyze GTP with the help of GAP (generally slow at hydrolysis without GAP)

Internal ribosome entry sites (IRES)

Sites in viral mRNA close to the AUG (start codon) that allow viruses to more easily and quickly initiate translation of their own mRNAs

Sometimes found in eukaryotic mRNA when the organism is under stressful conditions (due to cells having less ATP available)

eEFs

Eukaryotic elongation factors

Proteins that help with the translational elongation process

After the first amino acid is added to the tRNA, the rest of the amino acids are added by this

Load amino acids onto the tRNA (except for the first amino acid)

P (peptidyl) site

The middle location of the large subunit of the ribosome in which the first tRNA (with methionine) is plugged in

A (aminoacyl) site

The site of the ribosomal large subunit on the right side

The second location of where the tRNA with amino acids is plugged in

the large subunit of the ribosome between the P and A sites

Polypeptide bonds are formed in _______

Translocation

A process that occurs during translational elongation in which the ribosome moves the mRNA in groups of 3 nucleotides

This process is done by eEF2

E (exit) site

One of the 3 sites of the large ribosomal subunit in which the tRNA exits the ribosome and gets recycled

eIFs, eEFs, and release factors

The proteins that help with initiation, elongation, and termination are _______

eEF1By (beta)

A eukaryotic elongation factor and GEF responsible for the exchange of GDP to GTP

specific mRNAs; “globally” (at all mRNAs)

Translational regulation can occur at _______ OR __________

Autophagy

The cellular process of recycling

Ferritin

A protein that stores iron 

Only expressed (translated) when iron is present in the body

Iron response element (IRE)

A relatively short sequence of nucleotides located in the 5’ UTR of ferritin mRNA that is bound by an RNA binding protein

Iron response protein (IRP)

A protein responsible for turning off the translation of ferritin mRNA when iron is scarce or not present

Binds to the IRE, blocking the initiation of translation

RNA interference (RNAi)

A method of translational regulation through blocking gene expression by degrading specific mRNAs

MicroRNAs (miRNAs)

RNAs that repress translation via RNA interference (RNAi)

Each one of these RNAs targets ~100 different mRNAs

~ 1/3 human genes are regulated by this kind of RNA

One of these RNAs can degrade and regulate more than one mRNA, but at different degrees (perfect pairing or mismatched pairing)

Mismatched pairing 

The repression of translation that occurs due to imperfect pairing of nucleotides between miRNA and mRNA, causing a slower degradation of mRNA

Perfect pairing

The repression of translation that occurs due to exact pairing and perfect matches of nucleotides between miRNAs and mRNA, causing more efficient degradation of mRNA

Translational repressor proteins, RNAi with miRNAs, and regulated polyadenylation

Translational regulation at specific mRNAs includes ________, _________, and _________

Initiation factors eIF2 & eIF4E

Translational regulation “globally” at all mRNAs includes the regulation of ________

eIF2B

A GEF protein and a form of eIF2 that is bound to GDP after the transfer of methionyl tRNA into the ribosome

Exchanges GDP back to GTP

Can be regulated in order to stop translation generally —> can be inhibited by phosphorylation when cells are in stressful conditions (ex. absence of growth factors) 

Phosphorylation

The addition of a single phosphate to an element or protein

An allosteric interaction —> changes the conformation when it binds to a protein

4E Binding protein

A protein that interacts protein-protein with eIF4E

Acts as an inhibitor of translation —> blocks translation initiation by blocking eIF4E from being able to initiate translation (when the cell is in stressful conditions)

Can be phosphorylated when growth factors are present, inhibiting this protein and allowing translation to occur

Denaturing

The un-folding of a protein by heat or chemicals

After this occurs, proteins can be purified and re-fold into its original conformation due to amino acids being hydrophobic or hydrophilic

Chaperone proteins

Helper proteins that speed up the folding of proteins by pushing hydrophobic residues towards the insides of the protein

However, these proteins do NOT determine the 3D structure of a protein —> that is done by the actual amino acids in the sequence

Stabilize unfolded polypeptide chains to prevent aggregation during translation —> very important due to the high concentration of proteins in the cell and the potential for them to stick together when unfolded

Belong to the “heat shock protein” family because they are needed during heat shock to help with denatured proteins

Can help with the transport of proteins into organelles

Aggregation

The unfolding and clumping together of generally hydrophilic proteins creating hydrophobic deposits

Heat shock proteins

A family of proteins that includes chaperone proteins

Proteins that get over-expressed during heat shock

Chaperonins

A sub class of chaperone proteins that provide an isolated environment within which correct protein folding takes place

Create a barrel structure around the protein

Isolates the proteins from other proteins so that aggregation does not occur

ATP is involved with the movement in and out of the barrel structure —> regular chaperone proteins move the protein into the barrel

Alzheimer’s disease; Parkinson’s disease

2 diseases associated with the aggregation of misfolded proteins include _______ and _______

Amaloids

Aggregated B-sheet protein structures caused by the misfolding of proteins

Made by the misfolding and aggregating of proteins due to the exposure of lipophilic/ hydrophobic deposits leading to bigger and bigger aggregates that are no longer able to be degraded —> this leads to cell death because they are not able to be deposited —> often leads to disease

The initial misfolding that leads to Alzheimer’s disease

Tau

An aggregated protein associated with causing Alzheimer’s disease

Creates cognition problems in the brain

Aducanumab

The first successful drug created for treating Alzheimer’s disease

Acts as an antibody that targets B-sheet aggregates which commonly lead to diseases such as Alzheimer’s

Alzheimer’s disease

A disease caused by the increased aggregation of misfolded proteins with age and the decreased degradation mechanisms of these aggregates

APOE3 and reelin (RELN-COLBOS)

2 molecules that acts as protective variants against Alzheimer’s disease

Can help to delay the onset of Alzheimer’s by ~30 years

Protein Disulfide Isomerase (PDI)

An enzyme that acts as a chaperone by catalyzing protein folding

Involved in the formation, breakage, and rearrangement of disulfide bonds in nascent (newly synthesized) proteins

Makes an extra disulfide bond or takes away a disulfide bond in cysteine amino acids to ensure proper folding

Crucial enzyme in the ER

Peptidyl Prolyl Isomerase (PPI)

An enzyme that acts as a chaperone protein by catalyzing protein folding of proline amino acids

Helps proline go back and forth between different conformations (proline is the only amino acid that is restricted in its bond movement) 

Catalyzes the isomerization of peptide bonds in proline between the cis and trans conformations —> 90% of proline bonds are in the trans conformation, however cis conformation is often functionally significant

Proteolytic processing of insulin

The post-translational processing of insulin

The cutting of insulin from a sequence by a protease enzyme —> leads to the eventual degradation and regulation of glucose

A generally fast process because insulin is usually present —> the response to insulin is quick

Protease

An enzyme that cuts insulin out of a sequence

Becomes activated when insulin is available in the cell

Glycosylation

The post-translational modification process of adding carbohydrate chains to proteins in order to form glycoproteins

Allows for the protection and potential conformational changes of the protein

Plays and important role in protein folding in the ER, in targeting proteins for transport, and as recognition sites in cell-cell interactions

N-linkage glycosylation

A type of glycosylation that occurs on asparagine residues

Glycosylation starts in the ER

Formed by the addition of one sugar at a time 

O-linked glycosylation

A type of glycosylation that occurs on serine residues

Glycosylation starts in the golgi

Lipid addition to inner face plasma membrane proteins

A post-translational protein modification process in which lipid anchors are added to the protein allowing proteins to become associated with either the inner or outer layer of a phospholipid bilayer

N-myristoylation, prenylation, and palmitoylation

The 3 lipid anchors that allow proteins to attach to the inside of a phospholipid bilayer

GPI anchors

The type of lipid anchor that allows proteins to attach to the outer layer of a phospholipid bilayer

Phosphatase

An enzyme that removes phosphates generally transiently (temporarily)

Belong to 2 different families of proteins: serine/threonine and tyrosine