Mhg Lecture 6 - Translation

Front 1

Compare the stages of prokaryotic and eukaryotic translation.

Describe which proteins are specifically used for prokaryotes, which proteins are specifically used for eukaryotes, and which ones are the same.

Back 1

Front 2

What are genetic diseases of translation?

Back 2

-any mutation in the coding sequence is a genetic mutation of translation

-->there are silent mutations that may not have any effect, but if it’s a mutation that changes one amino acid to the other and it affects the function of the protein, you’re talking about a mutation affecting translation

-->can also affect the sequence or reading frame of the encoded protein.

Front 3

Where do most known disease causing mutations occur?

Back 3

Mutations that alter the mRNA coding sequence can lead to disease.


-Most known disease-causing mutations occur within the ORF

-Specific mutations affecting protein translation itself also occur

Front 4

What did scientist Paul Zamecnik do?

Back 4

Paul Zamecnik

-injected radioactive amino acids, isolated liver

- found radioactive proteins in ribonucleoprotein complexes

-no slide-see Lehninger


Lenhinger says:
In the early 1950s, Paul Zamecnik and his colleagues designed a set of experiments to investigate where in the cell proteins are synthesized. They injected radioactive amino acids into rats and, at different time intervals after the injection, removed the liver, homogenized it, fractionated the homogenate by centrifugation, and examined the subcellular fractions for the presence of radioactive protein. When hours or days were allowed to elapse after injection of the labeled amino acids, all the subcellular fractions contained labeled proteins. However, when only minutes had elapsed, labeled protein appeared only in a fraction containing small ribonucleoprotein particles. These particles, visible in animal tissues by electron microscopy, were therefore identified as the site of protein synthesis from amino acids, and later were named ribosome

Front 5

What did the scientific dream team of Zamecnik and Hoagland discover?

Back 5

Zamecnik and Hoagland

-found they could activate a heat-stable RNA (tRNA) by incubating with liver extracts (which coupled an amino acid onto the tRNA).

-No slide-see Lehninger


Lenhinger dice:
The second key advance was made by Mahlon Hoagland and Zamecnik, when they found that amino acids were "activated" when incubated with ATP and the cytosolic fraction of liver cells. The amino acids became attached to a heat-stable soluble RNA of the type that had been discovered and characterized by Robert Holley and later called transfer RNA (IRNA), to form aminoacyl-tRNAs. The enzymes that catalyze this process are the aminoacyl-tRNA synthetases

Front 6

Why should we remember the illustrious Francis Crick as a scientist, scholar, and a gentleman?

Back 6

Francis Crick

-same scientist who modeled B DNA

-reasoned that there must be some sort of RNA that could “translate” genetic code to amino acid sequence of a protein.

-No slide


Lenhinger says:
The third advance resulted from Francis Crick's reasoning on how the genetic information encoded in the 4-letter language of nucleic acids could be translated into the 20-letter language of proteins A small nucleic acid (perhaps RNA) could serve the role of an adaptor, one part of the adaptor molecule binding a specific amino acid and another part recognizing the nucleotide sequence encoding that amino acid into an mRNA (Fig. 27-2) This idea was soon verified. The IRNA adaptor "translates“ the nucleotide sequence of an mRNA into the amino acid sequence of a polypeptide. The overall process of mRNA-guided protein synthesis is often referred to simply as translation.

Front 7

Genetic code is a triplet non-overlapping code.

What does that even mean?
Who discovered this?
How did they do it?
Are there exceptions? Why might these exceptions exist?
Why is it in a triplet?

Back 7

(nobody knows what it means, but it's provocative .. it gets the people going)

Actually, Rosenthal says it means that there are three possible reading frames for each RNA
-It is a triplet NON-OVERLAPPING genetic code
-the genetic code turns the four letter alphabet of the DNA and RNA into the 20 amino acid alphabet of proteins
-it does this by reading triplets
-there are 3 different reading frames. The reading frame is usually set by a methionine starting the protein

-as with most scientific discoveries, it was an number of scientists over decades but the Nobel prize ultimately went to M. Nirenberg, P. Leder and H. Ghobind Khorana, who used complementary approaches to crack the genetic code 
-triplet binding studies allowed them to find which tRNA-aa’s bound to which triplets.


-There are a few exceptions to this, for example viruses will do what’s called a “stutter” so they’re read 3, 3, 3 and then step back 1 – gag pol of the retroviruses will do that – this is to economize a small amount of nucleic acids to try to get more out of them
-Even for human genes and E. coli genes there are a few exceptions to the non-overlapping rule, but in general it starts at the beginning and then reads 3, 3, 3 until it gets to the stop codon
-The reason that it’s 3 so you can get 64 possible codons.

Front 8

Who finally cracked the translational code? How did he do it?

Back 8

-Cracking of the code first by Marshall Nirenberg, who made RNA monomers (e.g. poly UUU), and added to extracts to synthesize monomer polypeptides (poly U makes poly phenylalanine, therefore UUU codes for phe).

Front 9

Who was able to figure out which tRNA-aa's bound to each triplet?

Back 9

M. Nirenberg and P. Leder

- triplet binding studies allowed them to find which tRNA-aa’s bound to which triplets.

Front 10

What did H. Ghobind Khorana do for molecular biology?

Back 10

H. Ghobind Khorana

-made synthetic RNA dimer, trimer and tetramers

Front 11

Where within the cell do the different steps of translation take place for eukaryotes? For prokaryotes?

Back 11

EUKARYOTES:
-mRNA is made in the nucleus and its spliced and polyadenylated and 5’ capped as it goes into the cytoplasm

-the translation takes place in the cytoplasm


PROKARYOTES:
-In prokaryotes where there is no nucleus you can have a coupling of transcription and translation and an opportunity of some forms of regulation as Dr. Martin talked about the transcription and translation of tryp and how you get different loops formed between 1, 2, 3, and 4. 
-That is NOT the same type of regulation you see in eukaryotes because it’s not coupled the same way

Front 12

What does the genetic code look like?

Back 12

-This is what the genetic code looks like
-All together there are 16 squares and in each square there are 4, so it’s every combination
-There are 64 potential codons of the mRNA and there are 20 amino acids

-So, looking at this you can see a couple of different things:

1. The genetic code is DEGENERATE
-->You can have multiple codons for one amino acid so the genetic code is DEGENERATE. 
-->That is NOT to say it’s ambiguous. If it’s being read on the ribosome and it sees CCC it will only put proline there. It WONT make a mistake
-->Mistakes ARE made 1/10,000 times in protein translation

2. They tend to cluster in different ways, most commonly in 2s or 3:1
-->in 2s: you have AA with a pyrimidine (U or C) will code for one amino acid and AA and a purine (A or G) will make another one
-->3:1 or all 4 – for example, UCA will be the same and G will be different

Front 13

How many possible codons are there in the triplet code?
How many amino acids does it need to code for?

Back 13

64 codons for 20 aa plus 3 stop codons = degenerate code.

Third position not as important (wobble hypothesis)

Front 14

True or False:
The genetic code is degenerate and ambiguous.

Back 14

1. The genetic code is DEGENERATE

64 codons for 20 aa plus 3 stop codons = degenerate code.

-->You can have multiple codons for one amino acid so the genetic code is DEGENERATE. 

-->That is NOT to say it’s ambiguous. If it’s being read on the ribosome and it sees CCC it will only put proline there. It WONT make a mistake

Front 15

How often are mistakes made in protein translation?

Back 15

Mistakes are made 1/10,000 times in protein translation

Front 16

At minimum, how many tRNAs are needed to recognize all of the codons?

Back 16

31 tRNAs necessary for all 20 amino acids
(16 squares x 2 tRNAs = 32 tRNAs
For stop codons, I don’t need a tRNA so 32 -1 = 31)

+

1 additional tRNA for initiator met
(because you need two tRNAs for methionine: 1 to begin the synthesis of the protein and one for internal methionines)

=

32 tRNAs minimum number needed to recognize all codons.


Recall that there are 64 codons for 20 aa plus 3 stop codons

Front 17

What's the anti-codon for AUC?

Back 17

The anticodon for AUC is GAU

-In the absence of other things, and it’s just 3 letters, always write from left to right 5’ to 3’

Note:
-This is what tRNA looks like with all the accessory proteins, initiation factors, elongation factors stripped away
-You have an mRNA codon being read from 5’ to 3’ and the anticodon pairing anti-parallel
-Whenever nucleic acids are pairing double stranded they’ll be anti-parallel

Front 18

How does one tRNA match more than one codon?

Back 18

WOBBLE HYPOTHESIS :)

-When you look at the whole chart there are 64 potential codons of the mRNA and there are three stop codons, at first glance you would think you need 61 anticodons or 61 tRNAs and in fact that’s not the case because you can have tRNAs matching more than one codon
(recall that only 32 tRNAs minimum number needed to recognize all codons)

-When they started sequencing tRNAs they realized that there are some modified ribonucleotides
-one called INOSINE is in the anticodon itself
-Inosine is the sugar + base (hypoxanthine)
-Hypoxanthine is an intermediate in the metabolism of nucleotides and ribonucleotides
-the anticodon is in the tRNA

Francis Crick came up with the WOBBLE HYPOTHESIS:
-He called the 3’ most of the triplet codon / the 5’ most of the anticodon the wobble position
-If you have an I in the anticodon wobble position it has the unusual property of being able to recognize A or U or C
-If this represents the anticodon, then you can see one tRNA recognizing 3 codons

Front 19

What happens when A or C is in the wobble position of the anticodon?

Back 19

A or C in wobble position of anticodon

--> only 1 codon recognized 
a. C:G
b. A:U

**there's only 1 AC ;)

Front 20

What happens when G or U is in the wobble position of the anticodon?

Back 20

G or U in wobble position of anticodon

--> 2 codons recognized 
a. G:C and G:U 
b. U:A and U:G

**AC goes to GU
(or conversely, at GU, AC goes to the coffee shop UG)

Front 21

What happens when inosine is in the wobble position of the anticodon?

Back 21

.I (inosine) in wobble position of anticodon

--> 3 codons recognized.
a. I:A and I:U, and I:C

Front 22

Can you have one tRNA that recognizes U, C, A and G?

Back 22

-You can’t have one tRNA that recognizes U, C, A, or G just because there is not one anticodon wobble position that recognizes all four, so you need at least two

--There are two ways to break this up:

1. Red box:
-If I match up A and A with a U and a U here and then I put a G here, I can match up both of these codons.
-It would read GUU from 5’ to 3’ and this would be UAA and that tRNA would put in lysine the one with GUU would put in asparagine and UUU in the anticodon would put in one for lysine

2. Pink box:
- In each box if all four are the same you can break it up the same way.
-In each box you can put two tRNAs
-You can’t do the same thing here because if I put in a U here there would be ambiguity, sometimes it would give me isoleucine and other times a methionine
-It would be confused here, the way you break it up is by putting an inosine here at the wobble position that recognizes UCA and here I’d have to put a C because otherwise it would create ambiguity (I thiiiink he’s talking about the blue box but I’m not positive :( )1

6 squares x 2 tRNAs = 32 tRNAs
For stop codons, I don’t need a tRNA so 32 -1 = 31+ 1 because you need two tRNAs for methionine: 1 to begin the synthesis of the protein and one for internal methionines
NEED A MINIMUM OF 32 tRNAs TO MATCH ALL THE CODONS

Front 23

Describe what happens in activation. What proteins are required?

Back 23

1. Requires aminoacyl-tRNA synthetase, ATP, specific tRNA, and specific amino acid that matches the tRNA

2. In a two-step reaction AMP is coupled to the amino acid with the release pyrophosphate (PPi), and then the amino acid is transferred to the 3’ OH of the 3’-most ribonucleotide of the tRNA (adenylate)-this activates the amino acid for peptide bond formation, and physically links the correct anticodon with its corresponding amino acid.
-PPi is hydrolyzed to inorganic phosphates (Pi), which means 2 phosphate bonds are hydrolyzed for each aminoacyl tRNA.

3. For two amino acids that resemble each other, a proofreading step is used on the aa-tRNA synthetase.
For example Valyl tRNA synthetase has a hydrolytic site that removes threonine if it mistakenly attached to AMP. This improves the accuracy of protein synthesis, which is about 1 error per 10,000 amino acids (but much less accurate than replication).

Front 24

What proteins are required for activation?

Back 24

1. Requires aminoacyl-tRNA synthetase, ATP, specific tRNA, and specific amino acid that matches the tRNA

-CCA is at the end after transcription ** Can Carry Aminoacids
-The amino acid is going to go on the 3’ A on the 3’ OH of this 3’ A always
-Here is the anticodon at this end, which pairs with the codon
-At the opposite end is where amino acids are going to link together to make the protein

Front 25

How do you activate the amino acid for peptide bond formation?

Back 25

2. In a two-step reaction AMP is coupled to the amino acid with the release pyrophosphate (PPi), and then the amino acid is transferred to the 3’ OH of the 3’-most ribonucleotide of the tRNA (adenylate)

Enzyme: amino acyl tRNA synthetase 
-There is one for each of the amino acids: eg. glycyl tRNA synthetase etc.
-Even if a tRNA looks different (for example, you can have different tRNAs for valine or leucine) it’s the same amino acyl that’s going to do it.
-In general, there are 20 amino acyl tRNA synthetases

-this activates the amino acid for peptide bond formation, and physically links the correct anticodon with its corresponding amino acid. 

-PPi is hydrolyzed to inorganic phosphates (Pi), which means 2 phosphate bonds are hydrolyzed for each aminoacyl tRNA.

-It uses ATP for energy. To put the amino acid on the very 3’ end it can’t do it directly because it takes too much energy so you have to give it energy and the energy currency in the cell is ATP or GTP.
**ATP -- tRNA Activation (charging)
**GTP -- tRNA Gripping and Going places (translocation)

-AMP from here is transferred onto the amino acid then its transferred from AMP onto the end of the tRNA and releases a pyrophosphate. 

-When you do the transfer, you hydrolyze between the alpha and beta phosphates and then as soon as the pyrophosphate is released it is hydrolyzed and drives the reaction forward by the law of mass action.

- When you remove product you drive the reaction forward.

-You’re sacrificing energy in order to get information.

Front 26

What does the final product of amino acyl tRNA synthetase look like?

Back 26

Enzyme: amino acyl tRNA synthetase 
-There is one for each of the amino acids: eg. glycyl tRNA synthetase etc.
-Even if a tRNA looks different (for example, you can have different tRNAs for valine or leucine) it’s the same amino acyl that’s going to do it.
-In general, there are 20 amino acyl tRNA synthetases


This is what the final product looks like
-This is NOT to scale
-This is the 4 leaf clover (the 2D structure NOT the inverted L)
-Then we’re going all the way to the last adenine and we have this the 3’ amino acyl group and depending on your R you’d have different amino acids
-Hybrid between nucleic acid and amino acid
-Note anticodon at bottom

Front 27

How does the amino acyl tRNA synthetase know how to take a tRNA on one hand and an amino acid on the other .. How does it know what amino acid to put on what tRNA?

Back 27

-There must be a second genetic code by which each synthetase recognizes each tRNA sequences. Deletion studies have shown that the ribonucleotides in the rectangles, and the anticodon are important in recognition.

-the orange boxes are the ones that are unique

-the anticodon is also important – it uses part of the regular genetic code in asking what the amino acyl tRNA synthetase sees about this that knows to put on valine or lysine

Front 28

What if two amino acids look like each other?

How do you ensure that it is not attached to the wrong tRNA?

Back 28

For two amino acids that resemble each other, a proofreading step is used on the aa-tRNA synthetase.

-balance between speed and fidelity
-we have evolved a way to get rid of threonine incase it tries to get hooked up

-For example Valyl tRNA synthetase has a hydrolytic site that removes threonine if it mistakenly attached to AMP.
-this is not going to happen with a weird looking amino acid like proline – there’s no proof reading activity there

-This improves the accuracy of protein synthesis, which is about 1 error per 10,000 amino acids (but much less accurate than replication).
-if you eliminate the proof reading activity the mistakes will be every once out of several hundred times (very common)

How does the proofreading work?
-this is the one that is supposed to see valine, it wants to see the hydrophobic methyl group from valine but instead threoinine is coming in
– it’s not matching but it went through long enough to get the AMP, to go through the first step. 
-It realizes it doesn’t match so it gets flipped to a hydrolytic site, an aminoacylation site (a proofreading site that specifically evolved to recognize the wrong amino acid)
-It flipped over and here is hydrolytic site and it chops the AMP off again.
-There is an energy cost for the sake of fidelity. 

- The pictures show what happens to prevent threonine from misincorporating onto the valine tRNA, but Lehninger refers to what happens to stop valine form misincorporating into isoleucine by a slightly different mechanism – Lehninger says they distinguish on basis of size. Not always just hydrophobic v. hydrophilic. Each one may use a different mechanism

Front 29

What are the components of the prokaryotic and eukaryotic ribosome?

Back 29

-there is a small subunit and a large subunit

-Both small and large subunits in both prokaryotes and eukaryotes have proteins and RNA
Eukaryotes: 40S + 60S --> 80S *Even
PrOkaryotes: 30S + 50S --> 70S *Odd

-S: how fast the settle in sucrose gradient. Be aware that the S of the two subunits does not equal the sum of each individual subunit
eg. 30+50 does not make 70, it’s how it sediments.

-the proteins are numbered S for small unit or L for large unit. They also have numbers, the same for prokaryotes and eukaryotes

-The numbering system is funny, some of the ones they thought were different were actually the same
--> this is actually 54 different proteins and this is around 82 different proteins, but basically the structure and function are similar for the ribosomes

-Guy called Nomura was able to figure all this out. He was able to reconstitute these things in a test tube from all the different proteins. 

-These are 18 and 23 nm – very large when they’re put together

-Small subunit has 1 RNA molecule

-the 18s and 16s are almost homologous/morphologous and have similar functions

-the 23s and the 28s have very similar functions

Front 30

What is the difference between a synthetase and a synthase?

Back 30

Synthetase uses ATP and a synthase does not have to use ATP

Front 31

What do you call the tRNA that accepts valine? What do you call it once it valine is attached?

Back 31

NOTE ON NOMENCLATURE

-The tRNA that accepts valine is called tRNA^Val.

-When valine is attached (or the tRNA is “charged”), it is called Val-tRNA^Val.

*Same rule applies for all amino acids*

Front 32

Describe the process of Initiation

Back 32

1. Components of Prokaryotic and Eukaryotic ribosomes

a. When we get to mutations in RPS19 and 24 that cause DBA, these are the proteins that we’re talking about, in the 40S subunit:
-In Prokaryotes mRNA binds to ribosomal small subunit (30S), which contains 16S RNA plus 21 different ribosomal proteins
- it is guided to the correct position by pairing of Shine-Dalgarno sequence in the 5’ end of the mRNA, and the complementary 3’ end of the 16S RNA
-Initiator methionyl tRNA (called tRNA^fMet in prokaryotes, tRNA^iMet in Eukaryotes) is bound to methionine by methionyl tRNA synthetase, then transformylase adds N-formyl group to the methionine group in prokaryotes
- the charged aminoacyl tRNA is now called fMet-tRNA^fMet. ** In eukaryotes, there is no formyl group, and it is called Met-tRNA^iMet.


4. fMet-tRNA^fMet is then bound to IF-2-GTP to allow binding of the fMet-tRNA^fMet to the P site of the small 30S ribosome, and the anticodon (usually CAU) pairs with the initiator codon of the mRNA (usually AUG codon).
NOTE ANTIPARALLEL PAIRING OF CODON AND ANTICODON. 


5. IF3 stops the 50S subunit from attaching too soon, while IF1 blocks the A site.


6. The large 50S subunit attaches to form the initiation complex, leading to the hydrolysis of GTP


7. In eukaryotes, the small 40S ribosomal subunit binds to the mRNA 5’ CAP. Met-tRNA^iMet is bound to the subunit with eIF2-GTP. eIF4F helps the complex scan the mRNA until the initiating AUG is found. Then joined by large 60S subunit, with hydrolysis of GTP and release of eiF2-GDP.

a. eIF2-GTP is regenerated by eiF2B-GTP. Mutations in eiF2B interfere with initiation and synthesis of myelin proteins- leads to VWM.

b. eIF2 can be phosphorylated and inhibited by PERK. Mutations in PERK lead to insulin overproduction, death of beta cells of pancreas, and therefore diabetes

Front 33

In prokaryotes, how is the RNA guided to the correct loaction?

How do the two ribosomal subunits fit together?

Back 33

In Prokaryotes mRNA binds to ribosomal small subunit (30S), which contains 16S RNA plus 21 different ribosomal proteins

- it is guided to the correct position by pairing of Shine-Dalgarno sequence in the 5’ end of the mRNA, and the complementary 3’ end of the 16S RNA

Left: prokaryotes
Right: eukaryotes

-The two subunits fit together like two gloves/ hands and the mRNA goes through the hands
-the tRNA is coupled to the amino acids and they burrow down in the cleft, the codons recognize the anticodons and that information is transferred via amino acid linked with a peptide bond.

Front 34

What does the small ribosomal subunit look like? What is it composed of?

Back 34

-this is a small subunit 

-the BROWN is the RNA and the BLUE is the protein

-even though there are a lot more proteins (21 in the small prokaryotic subunit) and only one ribosomal RNA in that 16s by bulk and by weight it’s taking up 2/3 of the whole ribosomal

-the rRNA does the lion’s share of the work of the ribosome

-the peptide bond itself is formed by the RNA component of the large subunit

-the 23s (prokaryotes) or the 28s (eukaryotes) actually catalyzes the peptide bond formation

– it’s a ribozyme, an RNA enzyme

Front 35

How do the initiator methionines compare between prokaryotes and eukaryotes?

Back 35

3. Initiator methionyl tRNA --> called tRNA^fMet in prokaryotes, tRNA^iMet in Eukaryotes

-The initiator methionyl tRNA is bound to methionine by methionyl tRNA synthetase, then transformylase adds N-formyl group to the methionine group in prokaryotes AFTER the methionine is coupled to the initiator tRNA
-The charged aminoacyl tRNA is now called fMet-tRNA^fMet.

-In eukaryotes, there is no formyl group, and it is called Met-tRNA^iMet.



Met-tRNA =
the sequence of the tRNA when methionine is coupled onto it in eukaryotes, methionine is the prefix:

–tRNA^fMet = the sequence of the tRNA of the initiator tRNA in prokaryotes, whether or not methionine is coupled onto that last 3’ A. This is just the tRNA sequence itself.
 ->if formyl-methionine is added onto the last A then it becomes fMet-tRNA^fMET

Front 36

Met-tRNA =

Back 36

the sequence of the tRNA when methionine is coupled onto it in eukaryotes, methionine is the prefix:

Front 37

–tRNA^fMet =

Back 37

the sequence of the tRNA of the initiator tRNA in prokaryotes, whether or not methionine is coupled onto that last 3’ A. This is just the tRNA sequence itself.

 ->if formyl-methionine is added onto the last A then it becomes fMet-tRNA^fMET

Front 38

Describe initiation in prokaryotes!!

Back 38

initiation

prokaryotes: **THIS IS DIFFERENT THAN THE MECHANISM IN EUKARYOTES
-the names are easy:
3 initiation factors: 1, 2, 3 – that is, IF1, IF2, IF3
release factors: 1, 2, 3



3 steps:

1. The small subunit couples to IF1 and IF3 and the mRNA to be translated

-the AUG is locked in place at the P site, how does it do that? ** remember, school STARTS in AUGust :)

-IF1:
-protects the A site, which is used for the 2nd through the last amino acid
-The P site is only used for the charged initiator methionine, the rest come into the A site

-IF3:
a. delays the attachment of the large subunit
-the purpose of IF3 is to keep the large subunit separate until the intermediate steps take place and then it allows the large subunit to come inb. helps to separate the large and small subunit after translation is completed


2. Add the initiator methionine, formyl-methionine hooked up to its correct tRNA (CAU)
-it’s hooked up to initiation factor 2 and that is hooked up to GTP
-IF2 brings the aminoacylated tRNA to the ribosome and attached it here


3. Large subunit comes on, hydrolysis of GTP --> GDP and then you have release of the Ifs
**ATP -- tRNA Activation (charging)
**GTP -- tRNA Gripping and Going places (translocation)

Front 39

How is the ribosomal/RNA structure held in place?

Why do you need GTP ?

Back 39

-it has 3D structure, needs to burrow down into the ribosome which takes energy

-this is held in place by three points of contact:
1. the codon with the anticodon
2. the tRNA, esp the T psi C arm is interacting with things in the large ribosomal subunit
3. How the the mRNA know to lock in place here?
There is a sequence here of 4-9 purines (A’s and G’s) located 8 to 13 bases upstream of the AUG, and that’s going to pair with the 16s ribosomal RNA in prokaryotes (see red lines!) and 18s in eukaryotes and it base pairs with this and if this is locked in place within the 16s in the small subunit and the mRNA binds to it, it locks the AUG in place at the P site.
That’s how AUG knows to go to the right site

remember,
**school STARTS in AUGust)
**ATP -- tRNA Activation (charging)
**GTP -- tRNA Gripping and Going places (translocation)

**THIS PROCESS IS DIFFERENT THAN THE MECHANISM IN EUKARYOTES

Front 40

What is a Shine-Dalgarno sequence?

Back 40

-This is a Shine-Dalgarno sequence

-that’s on the mRNA AG rich, just upstream of the AUG

-It base pairs with 16s in the small subunit and help lock it in place so the P site is open for the initiator methionine

-The Shine-Dalgarno sequence exists both in bacteria and archaea, being also present in some chloroplastic and mitochondial transcripts.

Front 41

Unfolded protein response:

Back 41

-The unfolded protein response (UPR) is a cellular stress response related to the endoplasmic reticulum. 

-The UPR is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum. In this scenario, the UPR has two primary aims: initially to restore normal function of the cell by halting protein translation and activate the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these objectives are not achieved within a certain time lapse or the disruption is prolonged, the UPR aims towards apoptosis.

Front 42

Describe initiation in Eukaryotes!!

Back 42

In eukaryotes, the small 40S ribosomal subunit binds to the mRNA 5’ CAP. Met-tRNA^iMet is bound to the subunit with eIF2-GTP. eIF4F helps the complex scan the mRNA until the initiating AUG is found. Then joined by large 60S subunit, with hydrolysis of GTP and release of eiF2-GDP.
a. eIF2-GTP is regenerated by eiF2B-GTP. Mutations in eiF2B interfere with initiation and synthesis of myelin proteins- leads to VWM.
b. eIF2 can be phosphorylated and inhibited by PERK. Mutations in PERK lead to insulin overproduction, death of beta cells of pancreas, and
therefore diabetes
__

-this is a picture from the review that he actually thinks anyone besides Paul is going to read

-there are different types of translation
eg. Cap dependent translation, which is the majority

-there are a number of different factors. Rather than locking in place directly like prokaryotes, this is the “bind and slide” : binds to the cap and slides to the first AUG. These factors here – the little e means eukaryotic – eIF2 is just like prokaryotic IF2, helps bring the Met in (**NOT formylated) the small subunit makes a complex with the cap and it has an RNA helicase

-they call it eIF4F and it has a helicase because RNA has all this secondary structure and it has to unwind it and slide this whole complex until it gets to AUG and then you can begin translation.

-Met is already in place and ready to go, then everything else is going to be similar

Front 43

How is initiation regulated in eukaryotes?

Back 43

With eukaryotes, you have REGULATION: 
-eIF2 is in the active form when its bound to GTP (just like in prokaryotes)
-But NOW you have other forms of regulation: ways of ACTIVATING and INACTIVATING it
-->Activating it: guanine nucleotide exchange factor called EIF2B 
-it is similar to the way that Ras works. Ras is active when it is bound to GTP when its hydrolyzed to GDP it’s no longer active so you have a guanine nucleotide exchange factor for Ras and for a lot of these other proteins that are involved that are oncogenes that are involved in signal transduction. It takes back the GDP and puts back the GTP and makes it active again. So when EIF2B is working, it keeps the initiation factor going and you have a lot of protein synthesis. 


Why would you need a lot of protein synthesis?

In most cases it’s fine and it just goes along at its normal rate, and that’s fine for the normal cell. But now say you have a cell that needs to make enough protein to ship out to other parts of the body, say like oligodendrocytes that have to make myelin to ship out to cover the main nerves of your body. It really has to churn it out at a high rate and keep this thing really active and initiation is most of the time the LIMITING FACTOR. You need to keep this churning out at a high rate. 

There is another one called PERK.
-They call it PERK because it’s an ENDOPLASMIC RETICULUM KINASE.
-If a protein is going to be shipped out, it has to be put in the endoplasmic reticulum. Anything that gets secreted is going to be put in the ER. It may be glycosylated or whatever but it’s ultimately going to be put in vesicles and secreted.
-PERK STOPS protein synthesis by phosphorylating eIF2. 


Why would you want to stop protein synthesis?

Say you have Beta cells in the islands of langerhans in the pancreas. It’s the same thing – insulin needs to be shipped out to other parts of your body so you have Beta cells making a lot of insulin and it’s being shipped out of the ER. Supposing it starts churning out too much insulin and it gets stuck in there and it gets UNFOLDED. Unfolded protein response, it says wait a second!!!!! You’re stuffing insulin in too fast!! Slow down until everything gets sorted out by chaperons and we degrade the unfolded proteins!!!It feeds back to PERK and shuts down protein synthesis, otherwise unfolded proteins just keep building up and you get something similar that you get in Alzheimer's or Parkinson's – too many unfolded proteins and the cell undergoes apoptosis.

IN EUKARYOTES:-these proteins are circled – if there are mutations in eIF2B, you might expect that protein synthesis might slow down. See the article. It’s a bit more complicated than that, it actually results in a deregulation of protein synthesis and you get an increase in the unfolded protein response. 
-If you have a mutation in PERK, you can’t shut down when there’s too much protein being churned out and there are no breaks. People who have this have a different genetic disorder.

Between prokaryotes and eukaryotes, elongation and termination are similar, just have different protein names, but initiation is different in terms of mechanism.

Front 44

Discuss the protein factors required for initiation of translation in bacterial and eukaryotic cells

Back 44

-the purpose of this chart is to match it up with the things he talked about

-he didn’t talk about eIF5 or eIF6.

-he did talk about eIF4 which is in the diagram. It’s A, E, and G that comprises the thing that “binds and slides”

-compare this to the diagram a few slides before-also talked about eIF2 and how it’s regulated in terms of vanishing white matter and Wolcott–Rallison Syndrome

-also talked about eIF2B and “these” .. Not sure which “these” are.

Front 45

Discuss elongation in prokaryotes and eukaryotes

Back 45

Prokaryotes:
3 elongation factors:

STEP 1:
-to go back we have the initiation complex with the initiator methionine – FORMYL met or else regular Met in eukaryotes – at the P site.
-All the aminoacyl tRNA is going to come in at the A site. So amino acid #2 is going to come in with help. This has 3D structure and it has to burrow its way down into this cleft, and in order to do that it needs energy and an elongation factor called Tu which is bound to GTP.
- Amino acid #2 burrows down and all the incoming ones in elongation go into the A site and it’s going to match the codon and the anticodon.

EF-Tu = elongation factor thermo unstable
-Just like in the initiation factor, the active form of EF-Tu is the GTP form.
- When it burrows down and locks in place there it hydrolyzes GTP to GDP. This is INACTIVE. In order to activate it, you get another guanine nucleotide exchange factor called EF-Ts which gets rid of the GDP and replaces it with GTP in these steps.
This is the 2nd elongation factor in prokaryotes. It gets rid of the GDP and GTP then replaces Ts and you’ve recycled it and its ready to attach to another amino acid.

C. Elongation

1. All new aa-tRNAs bind at the A site, coupled to EF-Tu (EF1alpha in eukaryotes) and GTP.
-After binding, GTP is hydrolyzed and EF-Tu-GTP is regenerated using EF-Ts (eEF1betagamma in eukaryotes).--> binding of aminoacylt-tRNA #2

2. Peptide bond formation is catalyzed by the 23S rRNA ribozyme (28S in eukaryotes) within the large 50S subunit. This is a nucleophilic attack of the carboxyl carbon of the previous amino acid by the electrons of the amino nitrogen of the incoming aa. The polypeptide grows from N terminus to C terminus as the ribosome complex reads the mRNA from 5’ to 3’.

3. EFG (eEF2 in eukaryotes), which mimics the structure of aa-tRNA-EFTu, binds to the A site, with the hydrolysis of a second molecule of GTP, and the translocation of the ribosome 1 codon (3 ribonucleotides) to the 3’ side of the mRNA. The uncharged tRNA is released from the E site. (In eukaryotes there is no E site and uncharged tRNA is released from P site).--> the whole ribosome shifts to the right!!! More likely, mRNA is going to shift to the left given the size of these subunits rather than the ribosome shifting to the right.

What shifts it?
We have ELONGATION FACTOR G!! This is called a translocase. It’s bound to GTP and it forces its way into the A site and the end result is now that it frees up the A site for the 3rd amino acyl tRNA. The one in the P site moves to E, A to P, and now there’s room for the next one to move down.

4. Steps 1-3 above are repeated until a stop codon is reached.

**No human inherited diseases of elongation factors are known. **

Front 46

In elongation, how is the peptide bond formation catalyzed?

Back 46

- Peptide bond formation is catalyzed by the 23S rRNA ribozyme in prokaryotes (28S in eukaryotes) within the large 50S subunit.

-This is a nucleophilic attack of the carboxyl carbon of the previous amino acid by the electrons of the amino nitrogen of the incoming aa.

-This makes the peptide bond!

-They call it a hybrid state in lehninger, it means that it’s leaning that way, crossing the A and P site.

-NOW you have a dipeptide on the 2nd tRNA.

-It’s getting transferred from the P site to the A site!! When the next ones come into the A site, two get transferred onto the new one and three get transferred onto the next one.

-The polypeptide grows from N terminus to C terminus as the ribosome complex reads the mRNA from 5’ to 3’.

__

NOW in eukaryotes:
-The same thing happens but the names are different
Instead of EF-Tu it’s called eEF1alpha
For Ts, the guanine nucleotide exchange factor, it’s called eEF1-beta-gamma

Front 47

How does a protein get into a site that is meant for tRNAs??

Back 47

-It’s strange when you think about it because EF-G and EF-Tu are proteins and they’re coming into the A site but the other ones are all tRNAs.


How does a protein get into a site that is meant for tRNAs??

-This is accomplished through molecular mimicry !!

- If you look at the structure of the charge and surface contour map, the bottom part is what normally comes in during the first part of elongation

-this is tRNA and the other is protein and it mimics the structure of that!!Fits into A site by mimicking the structure of tRNA!

Front 48

How do they get crystal structures of ribosomes?

What are the components of the 70S ribosome?

Back 48

Recall that
30S + 50S --> 70 S (Odd in prOkaryotes)
40S + 60S --> 80 S (Even in Eukaryotes)

-These are thousands of electron micrographs of ribosomes in vitreous ice – take in water and flash freeze so it doesn’t have time to form crystals, kind of like when you’re freezing cells and you don’t want crystals to form and rip the whole thing apart, so it’s a liquid the same way glass is a liquid so it doesn’t destroy it.

-Rather, it changes the structure of all 54 proteins, 3 rRNAs, 18 nm (prokaryotes)/23 nm (eukaryotes).

-That one protein changes the WHOLE structure.

-They think it functions as a ratchet, ratcheting the RNA right through one codon at a time every time GFP binds.

Front 49

What are the basic steps in termination?

Back 49

D. Termination

1. When a stop codon is reached, either RF1 or RF2 bind to the A site. RF1 recognizes stop codons UAA and UAG, while RF2 recognizes UAA and UGA

UGA = U Go Away
UAA = U Are Away
UAG = U Are Gone

2. The peptide is hydrolyzed from the last tRNA.

3. RF3 helps in the dissociation of the ribosome, accompanied by ATP hydrolysis.

**eRF3 mutations predispose to gastric cancer.**

Front 50

What are the basic steps in termination?

Back 50

D. Termination

1. When a stop codon is reached, either RF1 or RF2 bind to the A site. RF1 recognizes stop codons UAA and UAG, while RF2 recognizes UAA and UGA

2. The peptide is hydrolyzed from the last tRNA.

3. RF3 helps in the dissociation of the ribosome, accompanied by ATP hydrolysis.

**eRF3 mutations predispose to gastric cancer.**

Front 51

What happens in termination in prokaryotes?

Back 51

Termination: shows what happens in prokaryotes (will be similar for eukaryotes)


-Release factors: RF1, RF2, RF3

-There are 3 stop codons (UAA, UAG, UGA)

-RF1 recognizes UAA and UAG, while RF2 recognizes UAA and UGA

-RF3 helps RF1 and RF2 function properly

-The goal of all of this is to hydrolyze peptide and then process it and send it to wherever it needs to go and recycle the ribosome so that it can be used again on another mRNA

-Uses EFG to help translocate R or F ribosome recycling factor and IF3 (remember from beginning of lecture that IF3 stopped large subunit from coming on --> makes the large subunit dissociate and the end result is that everything is released)

Front 52

What happens in termination in eukaryotes?

Back 52

In eukaryotes, there is RF1 and RF3

-RF1 recognizes all 3 stop codons (humans don’t require 2 different release factors)
--> unusual situation where humans are simpler e. coli

- RF3 helps everything dissociate and hydrolyze the polypeptide

-In the book it says there is only 1 release factor but in the paper it says there are 2 (go with the paper)

-Unsure of all the steps in eukaryotes

-Some people will have mutations in RF3

Front 53

How do cells maximize their protein translating efficiency?

Back 53

-In order to be efficient and not have to wait for an mRNA to being fully translated before another protein can be made, when ribosomes move down a couple codons, a recycled ribosome will attach at another location and begin translating the mRNA too --> polysomes

-Can see this under EM where there will be multiple ribsomes on a single mRNA

Front 54

Inhibitors of initiation:

Back 54

Streptomycin (prokaryotes only)

Neomycin (prokaryotes only)

Front 55

Inhibitors of elongation:

Inhibitors that block peptidyl transferase

Back 55

Inhibitors that block peptidyl transferase:

1. cycloheximide (eukaryotes only)

Front 56

Inhibitors of elongation:

Inhibitors that act at A site and/or translocation

Back 56

Inhibitors that act at A site and/or translocation:


1. tetracycline (prokaryotes only)

2. erythromycin (prokaryotes only)

3. fusidic acid (prokaryotes only).

4. Diptheria toxin (eukaryotes only)
- ADP-ribosylates EF2 (eukaryotic version of EFG) to stop translocation.

5. Puromycin (prokaryotes and eukaryotes)
- mimics 3’ end of aa-tRNA and causes premature chain termination.

Front 57

Inhibitors of elongation:

Macrolide inhibitors

Back 57

Macrolide inhibitors are being tested to cause misreading of certain genetic mutations to “correct” the protein translation.