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 unit

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 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

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)

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

Also called eEF1By

GTPase activating protein (GAP)

Protein that accelerates the hydrolysis of GTP to GDP

GTP-binding turning off proteins 

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

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 processes of translation stops

Kinases

Enzymes that add phosphates

Include 2 substrates: amino acids and ATP

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

Docking site

Includes negatively and positively charged amino acids

Glycogen phosphorylase

Nuclear envelope

Made up of an inner and outer membrane (2 phospholipid bilayers and lumen)

Perinuclear space

the space in between the two membranes surrounding the nucleus

Continuous with the lumen of the ER

Nuclear lamina 

Made up of individual proteins called lamins 

Coiled coil dimer

Hutchinson-Gilford progeria syndrome

Emerin & LBR

Mutations can lead to progeria

Interact with each other through protein-protein interactions

Nuclear localization signals

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)

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 _______