PROTEIN SYNTHESIS IN BACTERIAL CELLS

Bacterias are prokaryotes and their cell does not contain any defined nucleus. Hence unlike the eukaryotes here all processes are carried on in the open cytoplasm.

Unlike the eukaryotic cell, the prokaryotic transcription and translation are carried on simultaneously and more rapidly.

Due to the rapidness, and the absence of governing systems these processes are often encountered with errors and mutations.

Usually, all bacterial cells contain a single chromosome in their cytoplasm without a nucleus. The chromosome contains the DNA that is the hard copy used as a template to make mRNA copies to produce proteins

Unlike eukaryotes, the mRNA is matured right at the sight of synthesis.

Transcription

The transcription is defined as the synthesis of mRNA from DNA that results in the transfer of information stored in from a certain portion of the heavy DNA (the gene) into a light mRNA.

A gene is referred to be the functional unit of DNA that is to be transcribed.

In a DNA bacterial cell, the DNA is used as the hard copy template to express the gene copy into mRNA. This is done by the enzyme DNA dependent RNA polymerase.

The figure of RNA polymerase (Fig-1) that has to have 5 units in its structure such as

two α units, β", β, and ω as follows:-

Fig-1

The β", β units are the first and second biggest units, followed by the two identical α units, which in turn followed by the ω (omega) subunit that is the smallest part in the enzyme structure. In addition to these, there is an initiating factor known as the sigma (ර) factor which helps the enzyme to initiate the process. All these six units are collectively known as holoenzymes.

The speed of the transcription is roughly 40 nucleotides per second.

TRANSCRIPTION (Prokaryotes)

The transcription occurs in four stages similar to eukaryotes.

1.Preinitiation

2.Initiation

3.Elongation

4.Termination.

1.Transcription-Preinitiation:

Transcription is selective and is carried on with a particular portion of the DNA that contains the necessary genetic message or a gene.

For transcription three basic things are necessary.

1.Template

2.Substrate

3.Enzymes.

1.Template:-

The template is the double-stranded DNA. The mRNA is synthesized by copying from the lower strand that is the noncoding strand or sense strand which run downstream to upstream (3' to 5')

The other upper strand is the coding strand or non-sense strand which runs in the opposite direction (5' to 3').

Fig-2

In the above figure, it is clear that the codons in the mRNA are always complimentary with the codons of the sense strand or non-coding strand and identical with the non-sense or coding strand (see Fig-2)

2.Substrates:-

1.ATP-Adenosine Tri Phosphate

2.GTP-Guanosine Triphosphate

3.CTP-Cytosine Triphosphate

4.UTP-Uridine Triphosphate

All the above are the RNA nucleoside triphosphates.

3.Enzymes:-

The required enzyme is DNA dependent RNA polymerase or RNAP. (see Fig-1 above)

In prokaryotes, a single RNA polymerase synthesizes all other RNAs such as mRNA, tRNA, rRNA, etc.

While in eukaryotes there are three RNAPs are functioning to produce the RNAs as follows:

RNAP-I -rRNA,and snRNA

RNAP-II -mRNA, and snRNA

RNAP-III -tRNA

The mitochondrial RNAP (mt RNAP) that transcribes all RNAs in the mitochondria.

The sigma factor always remains in separate factor and it fits with the core enzyme when the initiation begins as in Fig-3 below

Fig-3

The beta prime subunit is the largest followed by the beta subunit.

The two alpha subunits are having different terminal domains.

The alpha-1 subunit has an N- terminal domain which is associated with the assembly of RNA polymerase.

The alpha-2 subunit has a C-terminal domain which has a nonspecific interaction with the promoter on the DNA chain.

The omega subunit facilitates the RNA polymerase as well as stabilize the RNAP.

The sigma factor helps the RNAP to locate the promoter point of the transcription on the DNA template.

Fig-4

Fig-4A

In the above two figures how the promoter sites are located by the sigma factor has been illustrated as follows:-

As in the figure (Fig-4), there are two promoter sites are identified. The first site is the Pribnow box on the DNA pair base with the sequence T-A-T-A-A-T and is located -10 nucleotides from the start point. The other site with the sequence T-G-T-T-G-A-C-A. is located -35 nucleotides upstream from the start point. The start point is the +1 nucleotide of the gene to be transcribed.

Fig-4A shows the sigma factor locating the promoter site and help the RNAP to sit firmly on it.

2.Transcription-Initiation

The initiation of the transcription is subdivided into

1.Closed complex

2.Open complex

3.Abortive initiation.

Fig-5-The Closed Complex.

In the above Fig-5, the initiation started with the binding of the RNAP on the DNA at the promoter site located by the sigma factor (see Fig-4). This is the closed complex as the two DNA strands are not yet unwound to split apart.

Fig-5A-Open Complex & abortive-Initiation

In the above Fig-5A, it has been shown that the RNAP unwinds the DNA strand apart to form an open complex (The transcription bubble). In this stage, the RNAP initiates the synthesis of mRNA but only in short form because the presence of the sigma factor blocks the exit gate in the RNAP. The short mRNA formed helps the RNAP to be more stable. The next stage is the abortive initiation which departs the sigma factor away from the RNAP and opens the exit gate. (See Fig-5-A above)

3.Transcription-Elongation:-

Once the exit gate of the RNAP opened and the initiation is aborted by the departing of the sigma factor the RNAP moves on the template strand of the DNA towards the 5' to 3' direction continuously to synthesize the mRNA until to reach the termination point. New nucleotides are added at the OH- end of the 3'terminal to synthesize the mRNA in a 5'to3' direction. The elongated mRNA is exiting out and the process continues with at least 21 nucleotides (7 codons) or more with delayed termination. (See below The SUMMARY)

4.Transcription-Termination:-

There are two types of termination mechanisms. Both mechanisms are performed almost together.

1.Rho independent

2.Rho dependent. (ρ-protein)

1.Rho Independent:-(Intrinsic Termination)

In this process, the termination of mRNA without the involvement of a protein and is purely mRNA sequence-dependent. The codons at the terminal site decide the termination. This site is enriched with more Cytosines (Cs) and Guanines (Gs) that form complementary sequences of Gs and Cs in the mRNA. The Gs and Cs form strong triple bonds and attract each other to form a hairpin-like loop in the mRNA behind the RNAP.

As the RNAP moves towards the

5 to 3' downstream there are terminal signaling nucleotide codons such as T-A-A, T-A-G, T-G-A which are A-T rich. As a result, there are many uridine residues are formed at the tail end of the mRNA and many triple bonded G☰C (Guanine☰Cytosine) residues are formed at the other side of the RNAP when the RNAP reached the terminal codon. The strong triple bonded G☰C residues cause a hairpin-like twist in the mRNA. The hairpin-like twist cut of the mRNA from the weak double-bonded A=T rich terminal end of the DNA template + RNAP complex as shown in the Fig-6 below.

Fig-6

Rho (ρ) Protein Dependent Termination:

Fig-7

In this mechanism, the termination is performed by an ATP dependant protein called ρ (Rho) -protein. The protein is a helicase enzyme that can unwind the helical structure of DNA or DNA/RNA complex with the energy derived from the hydrolysis of ATP (Adenosine Triphosphate, a nucleoside triphosphate) to ADP.

The Rho protein follows a few nucleotides behind the RNAP during the elongation stage on the template strand. The cytosine rich area on mRNA attracts the hexamer, ρ (Rho) -protein to bind on the mRNA strand. When the RNAP reaches the terminal-sequence at the end of the gene, the RNAP stops to move. This may be due to the stop codons in front of the RNAP and the formation of a hairpin loop at the back of the RNAP. At this stage, the helicase enzyme gets the energy by the hydrolysis of ATP to ADP, runs fastly in a rotatory motion and catches the RNAP, and detaches the mRNA from the template strand-RNAP complex.

Generally, unlike human cells, the bacterial cells produce proteins rapidly in multiple systems with multi-RNAPs which can read on multiple genes simultaneously to produce multiple mRNAs or more elongated mRNAs at a time.

There are some proteins known as anti-stop proteins that are preventing termination of the transcription as follows:

1.This anti terminating proteins bind on the RNAP and signaling that not to stop at the stop sequence and to continue the template copying process.

2.Binding on the helicase ρ (Rho) -protein so that it cannot detach the mRNA from the DNA-RNAP complex.

3.Diffuse the formation of complimentary C-G rich sequences at the stop signal in the mRNA.

These anti-terminal proteins work in some compelling situations in which the cell needs more proteins to be made by delaying the termination.

PROKARYOTIC TRANSLATION

Unlike the eukaryotic or human cells, the transcriptions and translations are performed simultaneously in the cytosol itself in bacterial cells

Translation means protein synthesis as per the codon copied in the mRNA in the ribosomes.

The followings are the raw materials needed for the translation or protein synthesis:-

1.mRNA

2.tRNA

3.Ribosomes

4.The Protein Factors

5.Amino acids:-

There are 20 important amino acids are present in the human and as well as in the bacterial cell that can be carried by 20 amino acid-specific tRNAs.

6.Protein synthesizing or Translation is aided by Translation Factors (TFs). There are three types of Translation Factors. The first two factors are the Initiation Factors(IFs), and the Elongation Factors (EFs). Peptidyl transferase is an enzyme that is present in the 50S large subunit of the 70S ribosome helping to form the peptide bondage between the amino acids one by one.

The third one is the Termination Factors (RFs).-

Initiation of Translation:-

Similar to transcription, translation initiation also has 4 stages such as

1.Pre-initiation-tRNA charging

2.Initiation

3Elongation

4.Termination

1. Pre-initiation-tRNA charging

The initiation starts with tRNA Charging.

Prior to the initiation of the translation, the tRNAs should be charged with a suitable amino acid as shown below.

Fig-8-tRNA charging

As shown in the above figure (Fig-8) a tRNA is looking like a clover with four arms and a loop. The upper arm is the acyl (ACC) arm which can carry an amino acid.

The amino acid should be manipulated before attached to the tRNA as follows.

Amino acid + ATP ➝ (- 2PP-as Pyrophosphate)➝Amino acid-AMP

The amino acid AMP can now be bonded with the OH一 group of the ribose sugar moiety at the 3' end of the tRNA as shown in Fig-8. above. After the bondage, the AMP moiety is relieved out.

tRNA+aminoacd-AMP➝tRNA-amino acid + AMP

Now the tRNA is charged with the amino acid and carrying a corresponding anticodon at its foot. For example, if the amino acid is methionine then the corresponding anticodon is U-A-C to be carried by the tRNA at its foot.

2.Translation-Initiation.

Fig-9-A

The initiation is divided into two parts as shown in Fig-9 above.

1.30S initiation complex.

2.70S (or 50S) initiation complex

The "S" stands for svedbergs, a unit used to measure how fast molecules move in a centrifuge.

The 30s initiation complex is more important than the other and has several steps.

There are 3 initiation factors that play important roles in the 30S initiation complex are,

1.IF3

2.IF1

3.IF2

The above numbering is corresponding to their working order.

In the 30S smaller subunit there are three sites known as from left to right E (Exit site); P(Peptide binding site); and A (Accept site) (see Fig-9 above).

In the first step, the IF3 blocks the E-site. In the next step, the A site is blocked by IF1 followed by IF2. The IF2 is powered by ATP. With the blockade of E and A site, the P site is left free.

The ribosome is made of rRNAs and proteins. In the 16S rRNA, there is a sequence of nucleotides such as U-C-C-U-C which is known as the Shine Dalgarno sequence. The mRNA that has a complementary sequence such as A-G-G-A-G to this Shine Dalgarno (SD) sequence is attracted by the SD sequence in the 30S ribosome and attached to it (see Fig 9-A). The Shine Dalgarno sequence is located a few nucleotides away from the start codon AUG on the mRNA and the AUG is located exactly just below the P-site. (see Fig-9-A). The P-site is now free and open.

Fig-9-B

In the above figure, the first tRNA charged with a chemically modified methionine known as formyl methionine (fMet) enters the P-site directly and complements its anticodon U-A-C exactly with the start codon A-U-G on the mRNA. This is the end of the 30S initiation complex.

Then the IF3 released from the ribosome and the E-site is free to open. (see Fig-9-B)
The large 50S unit comes and fixes on the 30S unit to fit its three sites exactly with the sites of the 30S complex as shown in the Fig-9-C

Fig-9-C

After the fixing of the 50S unit, the IF1 followed by the IF2 is released from site A which eventually gets free and open. These are the events encountered in the 70S initiation complex.

Now all three sites A, P, and E are opened with the first tRNA sits in the P-site and matching its anticodon with the codon on the mRNA and enter into the next elongation stage.

Translation-Elongation:

The elongation process is carried out by three factors known as

1.EF1-Tu

2.EF2-Ts

3EF3-G

Fig-9-D

All three elongation factors mentioned above are GTP sensitive.

The end of the translation initiation is the 70S initiation complex in which the IF3, IF2, and IF1 are all moved away from the ribosome as in Fig-9D above.

After that, the elongation phase starts as in the Fig-10 below

Fig-10

The EF-Tu the first elongation factor starts the elongation by bringing a new charged tRNA with an amino acid with the respective anticodon and place it at the A site of the ribosome. (see Fig-10-A) below

Fig-10-A

After placing the tRNA at the A-site of the ribosome the EF-Tu-GTP becomes EF-Tu-GDP and returns to bring the next tRNA. Before that, it is recycled again to EF-Tu-GTP by EF-Ts. (see Fig-10-B)

Fig-10-B

Once the new tRNA is placed in the A site the peptidyl transferase an enzyme present in the ribosome makes a bond between the previous amino acid carried by the first tRNA at the P-site with the next amino acid carried by the new tRNA at the A-site. The ribosome moves one codon by the push given by EF-G-GTP

Fig-10-C

downstream to relocate at the next codon so that the charged tRNA slide into the P-site and the emptied first tRNA slides into the E-site to exit out.

Fig-10-D

The second new tRNA with its two amino acids slides into the P site pushed by the EF-G and read the codon on the mRNA at P-site.

The EF-G-GTP returns and becomes EF-G-GDP which then recycled to E-G-GTP by EF-Ts to continue to its next cycle.

Fig-10E

As the A-site is left free, another third charged tRNA is brought by the EF-Tu-GTP and placed at the A-site. Again the peptidyl transferase makes a bond between the previous two amino acids at the head of the previous tRNA at the P-site above the head of the new amino acid on the arm of the new tRNA at the A-site. Again the EF-G-GTP comes and gives a push to the tRNA at the A-site so that the 70S ribosome moves further one codon ahead towards 3' direction to relocate the tRNA from A site to the P site and the emptied previous tRNA already there at the P-site moves into the E-site so that the first tRNA that was already in the E-site is exited out. This is one complete Ribosome Cycle.

The cycles are repeated until a minimum of 22 molecules of amino acids are bonded to form a peptide chain.

Translation-Termination:-

This may be the last stage of the protein synthesis but unlike in the eukaryotic cells, bacterial protein synthesis is coupled and delayed to terminate the gene expressions to produce more and more proteins with the help of Termination Delaying Factors. This is undermined here and hence the normal translation termination is described here.

Fig-11

The termination phase starts when the ribosome encounters any one of the stop codons (UAA, UAG, and UGA) at the site-A. Stop codons cannot be complemented with normal tRNAs charged with amino acids. Hence at this stage special tRNA-like molecules either RF-1 or RF2 that have the complementary codons enter the ribosome at its A-site and complement the mRNA codons. In E-coli, the RF-1 and RF2 have the following complementary codons for the codons in mRNA in brackets.

RF-1:- AUU (UAA) & AUC (UAG)

RF-2:- AUU (UAA) & ACU (UGA)

When the mRNA stop codon is read by the RF-1 the peptidyl transferase enzyme is triggered by the exonuclease domain of the RF-1 to cleave out the entire peptide chain from the tRNA at the P-site (see Fig-11)

Fig-11-A

Now the RF3 comes into the 50S codon. Both RF-1 and RF3 are expelled out with the energy released from the conversion of GTP to GDP to which both of them are dependent. (see Fig 11-A)

Fig-11-B

After this, the elongation factor EF-G-GTP comes inside the A site of the 50S and give a push so that the ribosome moves one codon ahead down-stream

Add caption

towards the 3' of the mRNA as in the Fig-11-C below,

Fig-11-C

As the ribosome moves one codon 5' to 3' direction the uncharged tRNA in the E-site is expelled out and the EF-G-GTP becomes EF-G-GDP and exits out.

In the last step as in Fig-11-D below the initiation factor, IF3 comes into the 30S subunit as it does so both the subunits of the ribosome splits apart, the tRNA that is just uncharged in the P-site, and the mRNA are together expelled out. See Fig 11-D below:-

Fig-11-D

SUMMARY:-

Fig-12-A

Coupled Transcription & Translation

Fig-12-B

Unlike human beings, bacterias are unicellular organisms and need more proteins to be synthesized to survive.

Hence in their cells at a time many genes are expressed and the transcriptions and translations are coupled as simultaneous events. There are protein factors that help to prolong the transcriptions and translations by desensitizing the stop codons. There are multiple RNA polymerases to transcribe a prolonged gene expression. There are multiple ribosomes that translate at a time the mRNA to produce more proteins.