July 10, 2001 Lecture Notes

I. Introduction

The Central Dogma is

DNAà RNA à Protein

Translation is RNA à Protein

Translation involves the conversion of a nucleotide sequence (in triplet form) to an amino acid sequence.

II. There are four main players involved in translation:

mRNA

tRNA

ribosomes

accessory factors

In order to carry out translation efficiently, the cell needs protein factors to help with initiation, elongation, and termination.

A. mRNA

mRNA is a single stranded nucleic acid (most of the time).Translation proceeds from the 5’ end to the 3’ end. This means the N terminus of the protein is translated first, and the C terminus of the protein is translated last. Remember, not all mRNA is translated into protein — there are 3’ and 5’ untranslated regions (UTRs) that are important for regulation of translation.

B. tRNA

1.tRNA always has an unpaired 3’ end, and the sequence at the 3’ end of tRNA is always CCA.

2. tRNA has four arms, and is shaped like a very skinny clover leaf. The top arm of tRNA (the acceptor arm) is where the amino acid is attached. The amino acid is attached at the ribose of the 3’ adenosine via the hydroxy group. The amino acid is temporarily covalently attached to the tRNA (a dehydration reaction takes place).

*See Figure 5.3

3. tRNAs always have at least four arms, and some of them have an extra fifth arm. The most important arms to remember are the acceptor arm and the anticodon arm (which base pairs with mRNA). The structure of tRNA is relatively invariant.

*In the book anticodon sequences are denoted by a backwards arrow drawn over the top of the sequence.

*See Figure 5.2

4. The tRNA that is covalently attached to the amino acid is a charged tRNA (aminoacyl tRNA)

5. tRNAs have an extensive secondary and tertiary structure. They are shaped like an L with the amino acid on one end and the mRNA on the other end. This puts the amino acid and anticodon loop as far away from each other as possible.

*Note that Figure 5.4 is incorrect in that it has the amino acid bonded via the C, not by the carboxy group.

C. Ribosomes

1. Ribosomes are named according to how fast they sediment in a centrifuge tube. The unit of measurement is the Svedburg unit (S value).

2. Prokaryotic ribsomes are 70S, which can be broken down into 30S and 50S subunits. The 50S subunit is composed of 23S rRNA, 5S rRNA, and proteins. The 30S subunit is composed of 16S rRNA and proteins.

3. The small subunit binds to mRNA, so codon-anticodon base pairing takes place within the small subunit.

4. The large subunit has catalytic activity involved in forming the peptide bonds. Experiments tell us that the catalytic agent is rRNA.

5. Experiments also tell us about ribosome structure and function

a. Ribsomes were discovered in the 1950s.

b. Note that if you mix rRNA and ribosomal proteins in vitro, ribosomes will self-assemble.

c. Mutation analysis: If you mix mutant rRNA and ribosomal proteins in vitro, and run this against a control of wild type rRNA and ribosomal proteins, you can see which mutations in the rRNA halt ribosome assembly and/or function. This tells you that these bases are needed for ribosome assembly or function.

*See Figure 6.28

d. Protection Assay: This experiment tells you which regions on rRNA are protected by proteins. You can use nucleases or chemical probes (which are more useful because they are smaller) to cleave the rRNA, and run a gel of the fragments. No cleavage will occur where the proteins were protecting the rRNA, so there will be gaps in the gel at protein protection areas.

e. Chemical crosslinking: If rRNA and a protein are physically close enough together, reactive enzymes or chemicals will crosslink them.

f. X-ray crystallography: This allows you to look at the shape of the ribosome. When you combine this with mutation analysis it lets you see how a wild type and mutant ribosome are different in shape.

*See Figure 6.26

g. Proteins are important in maintaining the ribosome structure.

h. X-ray crystallography lets you look at ribosomes, tRNA, etc in certain configurations and in certain stages of translation. Antibiotics are especially useful in x-ray crystallography because they stop certain steps in translation, "freezing" the ribosomes in certain configurations. From this we know that there are many conformational changes in the ribosome during translation. Both subunits move relative to the mRNA and to each other.

6. Ribosome Structure

a. There are two sites for tRNA binding. The peptidyl site is called the P site and the acceptor site (which binds to the aminoacyl tRNA) is called the A site. tRNA binding involves regions in both the large and small subunits.

b. Relative to the mRNA, the P site is in the "Codon N" position while the A site is in the "Codon N + 1" position.

*See Figure 6.27

III. Translation in Prokaryotes (refer to handout)

Initiation includes everything up to the formation of the first peptide bond. During initiation the ribosomes assemble on the mRNA.

Elongation includes everything from the formation of the first peptide bond to the addition of the last amino acid. The ribosome moves down the mRNA by one codon each cycle (this is called translocation).

Termination is when release factors bind to the mRNA at the stop codon.

1. Initiation:

1.The small ribosome subunit binds to mRNA. This happens at the Shine-Dalgarno sequence — a purine rich sequence with a consensus of AGGAGG found about ten base pairs upstream of AUG. The 3’ end of the 16S rRNA base pairs with the Shine-Dalgarno sequence.

a. Note that experiments show that if there is a point mutation in the Shine-Dalgarno sequence, translation is decreased or inhibited. Mutations in the 16S rRNA at the 3’ end also inhibit or decrease translation. But, if you create both mutations in a complementary fashion (so they can still base pair with each other) then translation is restored.

2. The small subunit cannot bind to mRNA on its own. It needs the help of accessory factors. There are three initation factors (IF) in prokaryotes.

a. IF3 helps the 30S subunit bind mRNA. In addition, when IF3 is bound to the small subunit, the large subunit cannot bind to it. This makes IF3 a type of anti-association factor.

3. A special initiatior tRNA is used for initiation. In bacteria it is called tRNA^met _F . The methionine in this tRNA has been formylated (so the amino group is blocked and the tRNA can now not be used for elongation). This tRNA is also unique in that its bases before CCA in the acceptor arm are not base paired. This tRNA also has a GC stem loop in the anticodon arm. The GC loop is critical because it is needed for this tRNA to enter the partial P site. The charged initatior tRNA is written as met-tRNA_f .

*See Figure 6.9

The partial P site consists of the small subunit bound to mRNA.

4. The initiator tRNA needs an accessory factor to help it bind to the partial P site. IF2 is a monomeric G protein. A G protein can bind to GTP and GDP and has GTPase activity. G proteins are known as molecular switches, because their shape changes depending on whether they are bound to GDP or GTP. They are active when they are bound to GTP. IF2 forms a ternary complex of IF2, GTP, and the charged initiator tRNA. This ternary complex sets down the initiator tRNA in the partial P site.

5. Note that when the initiator tRNA binds to the partial P site, IF3 leaves the partial P site. When IF3 leaves this allows the 30S subunit to bind to the 50S subunit. This binding of ribosomal subunits stimulates the GTPase activity of IF2, causing it to cleave GTP to GDP. This causes IF2 to change its shape, and the IF2:GDP complex dissociates. The ribosome is now active.

6. IF1 helps form a more stable initiation complex.

*Figure 6.12 has the correct words but an incorrect picture.

B. Elongation

1.Every time a peptide bond is formed there is a cycle of elongation. The cycle is:

-aminoacyl tRNA binds to A site

-peptide bond forms

-ribosome translocates

2. There are three accessory factors involved: EF-Tu, EF-Ts, EF-G.

3. EF-Tu is in its active form when bound to GTP. It binds the aminoacyl tRNA, allowing it to bind to the A site.

4. Only aminoacyl tRNAs which can base pair to the codon are allowed to remain in the A site. Wrong tRNAs can enter, but they cannot remain at the A site.

5. When the codon and anticodon base pair, a conformational change in EF-Tu and the ribosome occurs. This triggers the GTPase activity of EF-Tu. The shape change caused by the GTPase activity results in the release of GDP. The hydrolysis of EF-Tu/GTP is very slow — because it is so slow, it allows time for an incorrect tRNA to dissociate.

*See Figure 6.19

6. The EF-Tu/GDP is regenerated by EF-Ts. The EF-Ts replaces the GDP, and is in turn replaced by GTP.

*See Figure 6.20

7. Peptide Bond Formation

Now we have the amino acid in the A site, covalently attached to tRNA and with a free amino group. We also have an amino acid in the P site, attached to the tRNA via a carboxy group. When EF-Tu leaves this causes the amino acids to be pushed close together. They are close enough so that a peptide bond can form, with the amino group in the A site attacking the C-O bond of the other amino acid/tRNA. This step is catalyzed by the 23S rRNA. During this bond formation the ribosome is stationary on the mRNA.

8. Translocation

This is tricky because the ribosome must move down one codon, but the tRNA must stay base paired to the mRNA. (See handout with hybrid states model) There is a transition state during translation when the acceptor arm of the P-site tRNA is in the E site of the ribosome, while the anticodon of the tRNA remains in the P site.

a. EF-G (a G protein) is used in translocation. It can only bind to the ribosome after EF-Tu has left, though, so perhaps they are competing for a similar binding site. When EF-G/GTP is hydrolyzed to EF-G/GDP, a shape change in EF-G occurs. This causes a shape change in the ribosome. The energy from the hydrolysis helps the ribosome move. The exit of EF-G is the last step in translocation, and can only occur when GTP has been cleaved.

*See Figure 6.24

C. Termination

1. Termination occurs at the UAG, UAA, or UGA stop codons. Release factors (RF1, RF2, and RF3) recognize stop codons, not tRNAs. Either RF1 or RF2 will bind to the A site, stimulating the hydrolysis of the peptidyl-tRNA bond. This results in a tRNA-OH and a protein with a carboxy terminus.

2. RF3 releases RF1 and RF2 from the ribosome. It is a G protein, and its GTPase activity allows the release of RF1 and RF2. Note that when it is bound to RF2 or RF1 it looks like EF-G —this illustrates conservation of tertiary structure.

3. The ribosome recycling factor (RRF) in conjunction with EFG binds to the A site of the ribosome. GTP hydrolysis causes dissociation of the ribosomal subunits from the mRNA and separates the two subunits from each other.

4. At this point IF3 can bind to the small subunit again to prevent the large subunit from reattaching.

IV. Eukaryotic Translation

A. The major difference from prokaryotic translation is that in eukaryotes the 5’ end of the mRNA is modified. There is a 5’-5’ linkage which attaches G to the 5’ end of the newly transcribed mRNA. This cap is important for mRNA stability and in translation. It is where the 30S subunit of the ribosome binds.

B. The 30S subunit moves along the mRNA until it gets to AUG.

*See Figure 5.17

C. Proteins involved in eukaryotic translation

1.eIF2 in eukaryotes is similar to IF2 in prokaryotes in that it helps a ternary complex of charged initiator tRNA, eIF2, and GTP to form.

Note that in eukaryotes the initiator tRNA methionine is not formylated.

2. eIF3 binds to the small subunit so it cannot be bound by the large subunit.

3. eIF4-4F is a multiprotein complex and has no prokaryotic counterpart. It binds to the 5’ end of mRNA.

4. eIF3 and eIF4 help the small subunit bind to the 5’ end of the mRNA. Proteins with ATPase activity (eIF4A) allow the unwinding of the secondary structure in mRNA.