Polypeptide Synthesis &

Ribosome Structure
Pre-Golgi, post-transcriptional protein

synthesis and the anatomy of a ribosome

Kyle Laracey
Wednesday, April 20th, 2011
Dr. Wachtmeister
 Introduction

Background Research

I. Ribosome Structure

The purpose of a ribosome is to translate the transcripted mRNA code into the

correct amino acids by means of amino-acylated tRNAs (1). Hence, this step of protein

synthesis is entitled translation.

The bacterial ribosome has an approximate mass of 2.6-2.8 MDa, a diameter of

200-250 Å, and a sedimentation coefficient of 70S (1). 70S represents the Svedberg

value, the rate of sedimentation in an ultracentrifuge. Svedberg units are not additive, and

thus the multiple subunits of a ribosome each have Svedberg values that may not add up

to the completed ribosome’s sedimentation coefficient (2). The 70S ribosome (found in

prokaryotes, e.g. E. coli) is comprised of two different sized subunits: the smaller 30S

subunit and the larger 50S subunit (1). In total, the 70S ribosome is made up of 3

ribosomal RNAs and 54 different proteins (3). The subunits are ribonucleoprotein

particles, each comprised of one third protein and two-thirds ribosomal RNA. The

smaller 30S subunit contains a single 16S rRNA; the larger 50S subunit contains both a

5S and 23S rRNA. Both the subunits can be described anthropomorphically. The 30S

subunit is said to have a “head” connected via a “neck” to the “body” of the subunit, with

a “shoulder” and “platform.” However, the 50S subunit is much more compact; it is

rounded and has only three protrusions (1). The peptidyl transferase center (the location

where peptide bonds are formed) is located wholly in the 50S subunit (1, 4).

A unique feature of the 50S subunit is the tunnel, which runs from the peptidyl-

transferase center (the site where peptide bonds are formed) right above the P-site on the

1
central protrusion to the base on the cytoplasmic face. Such a tunnel is also found in the

80S found in embryos of chicken. In fact, it is found in many ribosomes of radically

different species. The tunnel is one of many universally conserved features of the

ribosome, which are most likely vital to the functionality of the ribosome. It appears that

nascent proteins do indeed exit from this tunnel. (1, 5)

Because the tunnel is lined with hydrophilic, neutrally charged functional groups,

many kinds of nascent peptides may easily pass through. In fact, because protein

synthesis is such an exacting and complicated phenomenon, the components must

conserve their structure and ergo function throughout evolution. It is assumed that all

tRNAs, 16S and 23S (and alike rRNAs) all have identical overall secondary and tertiary

structures as well. (1)

Molecules of ribosomal RNA are produced in the nucleolus, which contains all

the genes corresponding to rRNA. The nucleolus is also one of the locations of the

assembly of ribosomal subunits from the ribosomal protein and rRNA. It is a type of non-

coding RNA and is a permanent component of the ribosomal subunits. rRNA primarily

functions in recognizing the different codons of mRNA and recognizing each tRNA. The

decoding center is located in the 30S subunit is constructed wholly of rRNA and

functions in lining up and positioning the mRNA and tRNA. The three tRNA binding

sites (A-, P-, and E- sites) of the ribosome are located on the 50S subunit and are

primarily comprised of rRNA. The PTC is also comprised of rRNA – specifically the 23S

strand. This leads many to consider the ribosome a ribozyme. (1, 6, 7)

In a similar fashion to the 50S tunnel, the rRNA of prokaryotic and eukaryotic

proteins are almost universally conserved, with the aforementioned 3 rRNA strands found

2
in all prokaryotic ribosomes, and a set of four rRNA strands found in all eukaryotes. This

is most probably because of the essential and complex role of rRNA in the ribosome. (8)

II. Structure of tRNA

tRNA is a fundamental part of protein synthesis. This is the delivery mechanism

for amino acids to the ribosome for assembly into a larger polypeptide (1). All tRNAs are

similar, each with 73-93 nucleotides. At the 3’ end of each tRNA is the nucleotide

sequence 5’ – CCA – 3’. This is the location that the amino acid binds to – i.e., the

Adenine of the CCA sequence. 7-15% of the bases of a tRNA are unique or modified; for

instance, certain adenines are changed to inosines. The secondary structure resembles that

of a cloverleaf (9, 10).

The tRNA has four loops: a D loop, an anticodon loop, a variable loop, and a T

loop (counter-clockwise) (1). Its tertiary structure resembles an uppercase “L” shape. The

anticodon loop and the CCA accepter stem are as far away from each other as possible –

at opposite ends of the tRNA. This is to ensure that there is no interference between the

anticodon of the tRNA and the codon of the mRNA by the acceptor stem’s CCA chain,

which contains no hydrogen bonds to other nucleotides of the tRNA (see figure). This

acceptor stem and the CCA chain is the docking site of the tRNA with the P- or A- site of

the ribosome (1, 10)

The amino acid is connected to the proper tRNA by means of a family of enzymes

called aminoacyl-tRNA synthetases. There is one aa-tRNA synthetase for each amino

acid. The accuracy of the aa-tRNA synthetases is absolutely crucial; there is no

3
proofreading done of the amino acid in the actual ribosome, only some concerning the

fidelity of the binding between the codon and a correct tRNA. (1, 10)

III. Initiation of protein translation

The actual synthesis of protein begins with the binding of the mRNA with the 30S

subunit. The ribosome having disassociated into its subunits or never having been

together in the first place, the “Shine and Dalgarno” (SD) sequence of the mRNA binds

with the anti-SD sequence on the 16S rRNA on the smaller ribosomal subunit (e.g. the

30S of the 70S, or the 40S of the 80S). The formation of the SD-anti-SD complex

primarily allows for the correct positioning of the start codon (AUG) of the mRNA at the

P-site. The SD sequence is to the left (i.e., towards the 5’ end) of the AUG start codon.

See Figure 1 (p. 10) for a diagram of the Shine Dalgarno sequence. (1, 9, 11)

After the formation of the SD complex, the special initiator tRNA charged with

formylated methione (called tRNAfmeti or fmet-tRNA) binds with the P site. In eubacteria,

initiation is catalyzed by three initiation factors: IF1, IF2, and IF3. Many more initiation

factors are required in eukaryotes/archaea. The mRNA and initiator-tRNAmeti in place,

this pre-initiation 30S complex binds with the 50S subunit, thus forming the 70S

ribosome. This connection is catalyzed by IF1. IF3 is believed to cause the 70S ribosome

to disassociate into its two subunits to begin the process. An overview of the initiation

stage may be seen in Figure 2 (p. 10). (1, 9, 11)

First, IF3 binds with the 30S subunit resulting in the splitting of the 70S ribosome.

IF3 positions the mRNA such that the start codon AUG is on the P-site. IF3 also

4
catalyzes the interaction of the anti-SD sequence and the 16S and 30S. Then, the fmet-

tRNA (with or without the help of IF2) binds at the P-site. (1)

IV. Elongation of nascent protein

The elongation cycle of a nascent protein can be divided into three phases: (i)

incoming animoacyl-tRNA occupation of the A-site. (ii) Peptide bond formation in which

the lengthening polypeptide chain is moved from the tRNA in the P-site onto the amino

acid of the A-site tRNA. (iii) Translocation, which is the movement of the mRNA bonded

to two tRNAs a distance of one codon. This moves the deacylated tRNA (i.e., the tRNA

which has just lost the polypeptide chain to the A-site tRNA) to the E-site (or exit site)

and the peptidyl-tRNA (tRNA previously occupying the A-site, charged with the nascent

polypeptide chain) to the P-site. See Figure 3 (p. 11) for a general orientation and layout

of the ribosome at the time of elongation. See Figure 4 (p. 12) for a detailed description

of elongation. (1, 9, 11)

Just as in initiation, elongation uses protein factors to catalyze the cycle. In

bacteria specifically, elongation factor (EF) G (EF-G) and elongation factor Tu (EF-Tu)

are very important for the speeding up of elongation. EF-G is involved in translocation,

and EF-Tu is involved in the positioning of the aa-tRNA into the A-site. Both Elongation

factors are GTPases. (8, 11, 12)

The cycle of elongation can also be divided into the PRE and POST states. PRE

refers to the ribosome before translocation, where the tRNAs are located in the P- and E-

sites, and POST refers to after Translocation, in which the tRNAs are located in the A-

and P-sites. The changing of the state of the ribosome (either POST  PRE or PRE

5
POST) is separated by a high activation-energy barrier of approximately 80-90kJ mol-1.

The elongation factors significantly reduce this barrier, thus significantly accelerating

protein synthesis more than 104 fold. In the absence of EF-G, spontaneous translocation

takes 2 minutes, while with EF-G, translation takes a mere 30 µs. “Spontaneous

translocation” refers to the phenomenon in which a ribosome will spontaneously change

from the PRE to POST state (after peptide bond formation, at 37 degrees Celsius, after

approximately 2 minutes). Thus, in this regard, EF-G is very similar to an enzyme. (1)

In stage one of elongation, the incoming aminoacyl-tRNA enters the A-site. This

aa-tRNA is bound with both EF-Tu and GTP, the source of the energy for the reaction

binding the codon and the cognate tRNA. The ternary complex aa - tRNA ∙ EF - Tu ∙ GTP

enters the A-site. EF-Tu hydrolyzes GTP to GDP, using the energy produced to bind the

aa-tRNA to the mRNA. The binary complex EF - Tu ∙ GDP is then released. (1, 11, 13)

In stage two, peptide transfer and translocation, the growing polypeptide located

on the peptidyl-tRNA located in the P-site is transferred onto the amino acid of the aa-

tRNA of the A-site (1). The peptydil-transferase center (PTC) is the location where the

peptide bond is actually formed, and is wholly contained in the larger 50S subunit (14,

15). The α-amino group of the aa-tRNA in the A-site of the PTC center “attacks” the

carbonyl group of the ester bond which links the peptidyl-tRNA in the P-site. The P-site

tRNA is thus made deacylated, and the growing polypeptide is then bound with the one

amino acid of the A-site tRNA by means of a peptide bond (1, 15).

Now, the deacylated tRNA of the P-site is to be translocated to the E-site, and the

new peptidyl-tRNA of the A-site must be translocated to the P-site. This translocation is

done with the aid of EF-G. This step results in the change of state of the ribosome from

6
the PRE to POST stage. Each tRNA will be moved precisely 10Å (the length of one

mRNA codon) to the left (towards the 5’ end). In itself, the ribosome has the ability to

perform translocation; however, EF-G functions to reduce the energy of activation,

thereby speeding up the reaction by a factor of 10. (1, 11, 13)

EF-G coupled with active GTP binds with the A-site peptidyl-tRNA. The

hydrolysis of GTP by EF-G causes translocation (10). A ratchet-like movement of the

smaller 30S subunit is induced. The deacylated tRNA moves into a hybrid P-E-site, and

the peptidyl tRNA is believed to be located between the A- and P-sites in a similar hybrid

position. The 30S ribosome then ratchets back into place, moving the two tRNAs along

into their proper positions simultaneously (1). The process of elongation continues until a

stop codon is encountered in the A-site (11).

V. Termination

In termination, once the ribosome reads a stop codon on the mRNA, the last

peptydil-tRNA is induced to release the polypeptide through the 50S subunit tunnel.

These codons are UAA, UAG, and UGA, none of which correspond to a tRNA. The

ribosome will then disassociate into its individual subunits. (1, 4, 13).

In prokaryotes, the stop codons UAA and UAG mentioned above are recognized

by release factor 1 (RF-1). UAA and UGA are recognized by RF-2. These two factors

bind in the A-site and induce the hydrolysis of the ester bond between the P-site tRNA

and the nascent polypeptide. The protein immediately is released and afterwards folds

into its proper tertiary level. RF3 (a GTPase) interacts with RF1 and RF2 and hydrolyzes

GTP to GDP as the energy for the hydrolysis of the ester bond. The energy released is

7
also used to move the mRNA along one codon, moving the last discharged tRNA into the

E-site, where it then leaves the ribosome. (10, 11)

The Experiment (16)

The goal in doing this experiment was to explore and observe protein synthesis in

action. With the help of the BU medical staff and CityLab’s supplies, an experiment

involving the synthesis of a protein called “GFP” was conducted. In it, a plasmid called

the “pGLO plasmid” would be inserted into the prokaryote E. coli (thus why the previous

research section concerned almost exclusively prokaryotic protein synthesis). This

plasmid would contain a sequence for GFP and an ampicillin resistance gene (to ensure

growth occurred). The GFP would be governed by an arabinose operon, which would

only work when the E. coli was in the presence of arabinose. The E. coli would be heat

shocked such that it would absorb the plasmid. This is described in steps 1 – 21 of

Transformation.

Six test plates containing plasmid-positive and plasmid-negative E. coli would be

created. Two would contain arabinose (ARA), LB broth (to help the E. coli recover from

the heat shocking undergone to insert the plasmid), and ampicillin. One of these two

cultures would not have the plasmid (it would be plasmid-negative). Another two plates

would have arabinose and LB broth but no ampicillin. One would be plasmid-positive,

and one would be plasmid negative. A final pair of plates would contain no arabinose, but

LB broth and ampicillin. One would contain plasmid-negative E. coli, and one would

contain plasmid-positive E. coli.

8
 As for predictions, the following should hold true. The ARA/LB/Amp plasmid-

positive plate should contain colonies and produce GFP because it contains the plasmid,

and contains ARA, thus, has ampicillin resistance and can produce GFP governed by the

arabinose operon because arabinose is present. However, as only about 5% of the E. coli

have been successfully transformed, only those 5% will live because they have ampicillin

resistance; thus, there will be colonies, not a lawn, because many will die. The

ARA/LB/Amp plasmid-negative plate should have no growth because without the

plasmid, the E. coli will die from ampicillin poisoning because they do not have the

proper gene to use to protect themselves.

The LB/ARA plasmid-positive plate as well as the LB/ARA plasmid-negative

plate should both have a lawn of E. coli, because there is no ampicillin to inhibit their

growth. GFP should be visible.

On the LB/Amp plasmid positive plate there should be growth similar to that on

the first plate mentioned above (ARA/LB/Amp plasmid+) because of the presence of

ampicillin and the ampicillin immunity gene. GFP should not be produced because of the

lack of arabinose, necessary for the arabinose operon governing the GFP gene to allow

the GFP gene to be transcribed and then translated into protein. In the LB/Amp plasmid

negative plate, there should be no growth because the E. coli would lack the ampicillin

resistance gene, and they were placed in ampicillin, inducing death.

9
Figure 4: Overview of elongation through the cycling of the PRE and POST stages (10)

10
List of Materials (16, 17)
- pGLO plasmid on ice, comprised of

o GFP gene

o Ampicillin resistance gene

- Arabinose

- Agar plates

- Sterile loops

- Incubator

- 1.5mL test tubes

- Foam racks

- P200 pipette

- P20 pipette

- Sterile tips

- Calcium Chloride (CaCl2) transformation solution

- Container of ice

- Starter plate of E. coli

- Hot water bath or incubator

- LB broth

- trysEDTA (TE) buffer solution

- dried lysozyme powder

- centrifuge

- 1.5 mL centrifuge tubes (what about the ones for transformation?

Procedure & Methods (16, 17)

11
Phase I: Transformation

1. Set the water bath or incubator to 42°C.

2. Label one closed 1.5mL test tube “(+)plasmid” and one “(-)plasmid”.

3. Label both tubes with your initials.

4. Place the tubes in the foam rack

5. Use a p200 micro pipette to transfer 200µL of the Calcium Chloride (CaCl2)

transformation solution into each 1.5mL test tube.

6. Place the tubes in the foam tube rack on ice.

7. Use a sterile loop to collect some E. coli bacteria from the starter plate – one

swab.

8. Immerse the swab with E. coli into the CaCl2 transformation solution in the

+plasmid test tube, towards the bottom.

9. Rotate the loop vigorously to dislodge the bacteria in the transformation solution.

10. Use a p200 micropipette to mix the solution.

11. Ensure there are no floating chunks

12. Use a new sterile loop and take one swab of the E. coli bacteria from the starter

plate.

13. Immerse the swab of E. coli into the CaCl2 transformation solution in the

(-)plasmid test tube towards the bottom.

14. Place the (-)plasmid test tube on the ice.

15. Retrieve the pGLO test tube from the ice bucket.

16. Retrieve the (+)plasmid test tube from the ice.

12
17. Use a p20 micropipette to extract 10µL of pGLO plasmid from the pGLO test

tube.

18. Add the 10µL of pGLO plasmid to the (+)plasmid test tube

19. Return the (+)plasmid to the ice.

20. Return the pGLO test tube to the ice bucket

21. Allow the (+)plasmid and (-)plasmid test tubes to cool on the ice for 10-15

minutes.

22. While the tubes are cooling on the ice, label the six agar plates on the bottom as

follows for each individual plate (a – f):

a. (+)plasmid; LB; Amp; ARA;

b. (+)plasmid; LB; ARA;

c. (+)plasmid: LB; Amp;

d. (-)plasmid; LB; Amp; ARA;

e. (-)plasmid; LB; ARA;

f. (-)plasmid; LB; Amp;

23. Write your initials and the date on the bottom edge of each plate.

24. Bring the ice bucket with the (+)plasmid and (-)plasmid to the water bath or

incubator

25. After the tubes have been on ice for 15 minutes, transfer both tubes to the water

bath or incubator while the tubes are in the foam rack, keeping them afloat (if in

the water bath) or standing up (in the incubator).

26. Ensure to push the tubes all the way down in the rack so the bottoms of the tubes

make contact with the warm water (if using a water bath).

13
27. Allow them to sit in the water bath or incubator for exactly 50 seconds.

28. After the 50 second incubation, transfer both test tubes in the foam rack back onto

the ice.

29. Allow them to cool on the ice for 2 minutes.

30. During the 2 minutes of cooling, set the incubator or water bath to 37°C.

31. After 2 minutes, remove the tubes from the ice.

32. Remove the (+)plasmid tube from the foam rack.

33. Open the (+)plasmid tube.

34. Use a p200 with a sterile tip to add 200µL of LB broth to the (+)plasmid tube.

35. Eject the sterile tip into the contaminated bin.

36. Close the (+)plasmid tube.

37. Place the (+)plasmid tube back on the foam rack.

38. Remove the (-)plasmid tube from the foam rack.

39. Open the (-)plasmid tube.

40. Use a p200 with a sterile tip to add 200µL of LB broth to the (-)plasmid tube.

41. Eject the sterile tip into the contaminated bin.

42. Close the (-)plasmid tube.

43. Place the (-)plasmid tube back on the foam rack.

44. Place the foam rack in the water bath or incubator.

45. Allow the tubes to heat for 10 minutes.

46. After 10 minutes, mix the contents of each tube by tapping your finger against the

side of the test tube.

14
 47. Use a p200 micropipette with a sterile tip to add 100µL of bacteria from the

(+)plasmid tube to each plate labeled (+)plasmid.

48. Dispose of the sterile tip.

49. Use a p200 micropipette with a new sterile tip to add 100µL of bacteria from the

(-)plasmid tube to each plate labeled (-)plasmid.

50. Dispose of the sterile tip.

51. Use a sterile loop to spread the bacterial suspensions placed on the agar plates

evenly around the surface of the agar by gently dragging the flat surface of the

new loop back and forth across the plate’s surface.

52. Ensure not to mark up the agar too much; do not press on the agar to much,

making it appear scarred.

53. Place the plates upside down in an incubator at 37°C for 24 hours or not in an

incubator at room temperature for 2-3 days.

Phase II: Cell Lysis and GFP Extraction

1. Add 250µL of TE buffer solution to a 1.5mL microcentrifuge tube.

2. Label this tube “AC Lysozyme”.

3. Transfer 3 swabs of bacteria with a sterile loop from the agar plate containing

fluorescent E. coli into this tube.

4. Resuspend the bacteria by putting the tube in the vortex machine, or by repeatedly

extracting solution with a micropipette and ejecting the solution back into the

tube.

5. Uncap the AC Lysozyme tube.

15
 6. Add 55µL of lysozyme to the AC Lysozyme tube.

7. Recap the AC Lysozyme tube.

8. Mix the contents of the AC Lysozyme tube by flicking the tube with the index

finger.

9. Place the sample on dry ice for 5 minutes.

10. Thaw the sample by placing the tube in the palm of the hand. Body heat is enough

to thaw the sample.

11. After the sample is thawed, place the sample opposite a sample of equal

weight/contents in a centrifuge.

12. Centrifuge the two samples for 2 minutes at 2000RPMs.

Observations & Results

At the lab, a specimen of GFP producing E. coli had already been produced over

the weekend. It had been sitting for approximately two days at room temperature. When a

UV light was shined on the E. coli, it appeared green, thus indicating that GFP had

indeed been produced. The petri dish clearly showed signs of growth. The green,

however, was hard to see, a light shade of green.

Once the E. coli cells had been lysed and the GFP isolated in a crude extraction,

the GFP could clearly be seen. Under UV light, the bottom of the test tube appeared

extremely green. GFP had been created and isolated.

See Figures 21 – 22 (p. 37 – 38) for evidence of the GFP isolated.

At home, there was growth on certain plates. However, very little to no GFP

could easily be seen. In the plate with a plasmid, LB broth, ampicillin, and arabinose (+,

16
LB, AMP, ARA) extreme growth was observed. There was very little food left. The small

splash of food (whitish) can readily be seen from the images. No GFP or green was seen

under UV light. In the corresponding plate without a plasmid, with LB broth, ampicillin,

and arabinose (-, LB, AMP, ARA), no growth was detected. No GFP was detected. In the

plate with a plasmid, LB broth, ampicillin, but no arabinose, growth was detected. There

was no GFP or green to be found though. In the corresponding plasmid-negative plate

with LB broth and ampicillin, neither growth nor green/GFP was detected. In the plate

with a plasmid, LB broth, arabinose, but no ampicillin, growth was detected but no green

or GFP. In the corresponding plasmid-negative plate with LB broth, arabinose, but no

ampicillin, Growth was detected, but without green/GFP.

It is difficult to discern growth from the pictures taken, but in person, growth was

clearly visible on all those stated above to have growth, and none visible on those stated

above to be growth-less.

There was a pungent smell in the air after opening the bag containing the plates of

E. coli.

See Figure 5 (p. 21) for the appearance of the plates before the E. coli had been

sitting for several days. See Figures 6 – 14 (p. 22-30) for pictures of the plates after the

E. coli were allowed to grow, and Figures 15 – 20 (p. 31 – 36) for images of the plates

under UV light.

17
 Conclusion & Analysis

At the lab, the experiment was a success. Working from pre-grown E. coli

containing the GFP plasmid, which had already produced GFP, the extraction and

isolation of that GFP was a success. The most probable explanation for why the GFP in

the petri dish appeared a lighter green color than that of the concentrate found in the test

tube is that the cell membrane and cell wall obstruct the view of the innards of the E. coli,

thus, the GFP is not as easily seen.

At home, the primary issue was that there was little to no GFP observed. This

may have been because the E. coli on the plates were allowed to grow for too long; five

days afterwards, the plates were checked on. This would explain the extreme growth in

the first plate and lack of agar (they were given ample time to consume all the food, and

then die because of starvation). None of the plates experienced GFP glowing under UV

radiation (see figures 15 – 19), contrary to the results predicted.

References

1. Nierhaus KH, Wilson DN, editors. 2004. Protein Synthesis and Ribosome Structure.
Germany: Wiley-VCH.

2. Davidson MW, Florida State University. 2005. Ribosomes. [online]

http://micro.magnet.fsu.edu/cells/ribosomes/ribosomes.html. (Last accessed April 18,
2011).

3. Max Planck Institute for Molecular Genetics. 2001. Ribosomal structure and
ribosome-antibiotic interactions. [online]
http://www.molgen.mpg.de/~ag_ribo/ag_franceschi/franceschi-main.html. (Last
accessed April 18, 2011).

4. St. Edward’s University. 1995. Ribosomes. [online]

http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/Ribosomes/Ribos
ome.HTML. (Last accessed April 18, 2011).

18
5. Voss NR, Gerstein M, Steitz TA, Moore PB. 2006. The Geometry of the Ribosomal
Polypeptide Exit Tunnel. [online] http://papers.gersteinlab.org/e-
print/ribotunnel/preprint.pdf. (Last accessed April 18, 2011).

6. Encyclopædia Britannica. 2011. Ribosomal RNA (rRNA).

http://www.britannica.com/EBchecked/topic/502158/ribosomal-RNA-rRNA. (Last
accessed April 18, 2011).

7. UCSF McManus Lab. 2009. Ribosomal RNA. [online]

http://mcmanuslab.ucsf.edu/node/260. (Last accessed April 18, 2011).

8. New World Encyclopedia. 2008. Ribosomal RNA. [online]

http://www.newworldencyclopedia.org/entry/RRNA. (Last accessed April 18, 2011).

9. King MW, IU School of Medicine. 2011. Protein Synthesis. [online]

http://themedicalbiochemistrypage.org/protein-synthesis. (Last updated March 24,
2011).

10. Luecke H. 2011. tRNA Structure. [online]

http://bass.bio.uci.edu/~hudel/bs99a/lecture21/lecture2_2.html. (Last accessed April
18, 2011).

11. The Royal Swedish Academy of Sciences. 2009. Structure and Function of the
Ribosome. [online]
http://nobelprize.org/nobel_prizes/chemistry/laureates/2009/cheadv09.pdf. (Last
accessed April 18, 2011).

12. Campbell NA, Reece JB. 2011. Biology. 9th ed. San Francisco (CA): Pearson
Education, Inc. 1263p.

13. Kimball JW. 2010. Gene Translation: RNA -> Protein. [online]
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Translation.html. (Last
updated April 22, 2010).

14. Encylcopædia Britannica. 2011. Peptidyl Transferase. [online]

http://www.britannica.com/EBchecked/topic/450922/peptidyl-transferase. (Last
accessed April 18, 2011).

15. Moore PB, Steitz TA. 2009. After the ribosome structures: How does peptidyl
transferase work? [online] http://rnajournal.cshlp.org/content/9/2/155.full.pdf . (Last
accessed April 18, 2011).

16. Boston University Medical Center. 2011. Transformation – Procedure. 7p.

17. Boston University Medical Center, Trustees of Boston University. 2004. Cell Lysis
Protocols for the Protein Extraction Station. 9p.

19