15.  Elaborate how proteins' initiation, elongation, and termination happen in the ribosome with suitable diagrams.

answer :

Introduction :

Ever wonder how antibiotics kill bacteria—for instance, when you have a sinus infection? Different antibiotics work in different ways, but some attack a very basic process in bacterial cells: they knock out the ability to make new proteins.

To use a little molecular biology vocab, these antibiotics block translation. In the process of translation, a cell reads information from a molecule called a messenger RNA (mRNA) and uses this information to build a protein. Translation is happening constantly in a normal bacterial cell, just like it is in most of the cells of your body, and it's key to keeping you (and your bacterial "visitors") alive.

When you take certain antibiotics (e.g., erythromycin), the antibiotic molecule will latch onto key translation molecules inside of bacterial cells and basically "stall" them. With no way to make proteins, the bacteria will stop functioning and, eventually, die. That's why infections clear up when they're treated with the antibiotic.$^{1,2}$start superscript, 1, comma, 2, end superscript

Cells need translation to stay alive, and understanding how it works (so we can shut it down with antibiotics) can save us from bacterial infections. Let's take a closer look at how translation happens, from the first step to the final product.

Translation: Beginning, middle, and end :

A book or movie has three basic parts: a beginning, middle, and end. Translation has pretty much the same three parts, but they have fancier names: initiation, elongation, and termination.

• Initiation ("beginning"): in this stage, the ribosome gets together with the mRNA and the first tRNA so translation can begin.
• Elongation ("middle"): in this stage, amino acids are brought to the ribosome by tRNAs and linked together to form a chain.
• Termination ("end"): in the last stage, the finished polypeptide is released to go and do its job in the cell.

Initiation :

In order for translation to start, we need a few key ingredients. These include:

• A ribosome (which comes in two pieces, large and small)
• An mRNA with instructions for the protein we'll build
• An "initiator" tRNA carrying the first amino acid in the protein, which is almost always methionine (Met)

During initiation, these pieces must come together in just the right way. Together, they form the initiation complex, the molecular setup needed to start making a new protein.

Inside your cells (and the cells of other eukaryotes), translation initiation goes like this: first, the tRNA carrying methionine attaches to the small ribosomal subunit. Together, they bind to the 5' end of the mRNA by recognizing the 5' GTP cap (added during processing in the nucleus). Then, they "walk" along the mRNA in the 3' direction, stopping when they reach the start codon (often, but not always, the first AUG).$^6$start superscript, 6, end superscript

Eukaryotic translation initiation:

1. Complex of small ribosomal subnit and initiator tRNA (bearing methionine) binds to 5' cap of mRNA.
2. Complex scans from 5' to 3' to find the start codon (AUG).
3. Initiator tRNA binds to start codon.
4. Large ribosomal subunit comes together with the mRNA, initiator tRNA, and small ribosomal subunit to form the initiation complex. The initiator tRNA is positioned in the P site of the assembled ribosome.

These steps are assisted by initiation factors (not shown in diagram).

Based on similar diagram in Berg et al.$^1$start superscript, 1, end superscript

In bacteria, the situation is a little different. Here, the small ribosomal subunit doesn't start at the 5' end of the mRNA and travel toward the 3' end. Instead, it attaches directly to certain sequences in the mRNA. These Shine-Dalgarno sequences come just before start codons and "point them out" to the ribosome.

Bacterial translation initiation:

On a bacterial mRNA, a G/A-rich sequence called the Shine-Dalgarno sequence is found slightly upstream (5' of) the start codon (AUG). The small ribosomal subunit recognizes and binds to the Shine-Dalgarno sequence. The small ribosomal subunit also binds to the initiator tRNA (carrying fMet), which forms complementary base pairs with the start codon. As noted in the text, the small ribosomal subunit may sometimes bind first to the mRNA (and then the initiator tRNA), and sometimes the other way around (the initiator tRNA first, and then the mRNA). It's thought that the order of these events may be random.

Once these components have come together, the large ribosomal subunit joins them. The assembled ribosome with mRNA and bound initiator tRNA comprises the initiation complex. The initiator tRNA is in the P site of the assembled ribosome.

Bacteria use fMet (a chemically modified methionine) as the first amino acid.

Why use Shine-Dalgarno sequences? Bacterial genes are often transcribed in groups (called operons), so one bacterial mRNA can contain the coding sequences for several genes. A Shine-Dalgarno sequence marks the start of each coding sequence, letting the ribosome find the right start codon for each gene.

Eukaryotic cell:

• DNA is transcribed to make an RNA inside the nucleus. The initial RNA transcript is processed into a mature mRNA before exportation to the cytosol.
• The mRNA contains just one coding sequence (specifying one polypeptide).

Bacterial cell:

• DNA is transcribed to make an mRNA in the cytosol. Ribosomes can start translating the mRNA before it is even completely transcribed. No post-transcription processing steps are necessary.
• The mRNA contains three coding sequences from three different genes, each specifying its own polypeptide.

Elongation :

I like to remember what happens in this "middle" stage of translation by its handy name: elongation is when the polypeptide chain gets longer.

But how does the chain actually grow? To find out, let's take a look at the first round of elongation—after the initiation complex has formed, but before any amino acids have been linked to make a chain.

Our first, methionine-carrying tRNA starts out in the middle slot of the ribosome, called the P site. Next to it, a fresh codon is exposed in another slot, called the A site. The A site will be the "landing site" for the next tRNA, one whose anticodon is a perfect (complementary) match for the exposed codon.

In the first round of elongation, an incoming amino acid attaches to methionine already present in the ribosome's P site. This action initiates the growth of a polypeptide. The three steps of this first round of elongation are described below.

1) Codon recognition: an incoming tRNA with an anticodon that is complementary to the codon exposed in the A site binds to the mRNA. Energy from GTP is expended to increase the accuracy of codon recognition.

2) Peptide bond formation: a peptide bond is formed between the incoming amino acid (carried by a tRNA in the A site) and methionine (a tRNA charged with methionine attached to the P site during initiation). This action passes the polypeptide (the two bonded amino acids) from the tRNA in the P site to the tRNA in the A site. The tRNA in the P site is now "empty" because it does not hold the polypeptide.

3) Translocation: the ribosome moves one codon over on the mRNA toward the 3' end. This shifts the tRNA in the A site to the P site, and the tRNA in the P site to the E site. The empty tRNA in the E site then exits the ribosome.

Once the matching tRNA has landed in the A site, it's time for the action: that is, the formation of the peptide bond that connects one amino acid to another. This step transfers the methionine from the first tRNA onto the amino acid of the second tRNA in the A site.

Not bad—we now have two amino acids, a (very tiny) polypeptide! The methionine forms the N-terminus of the polypeptide, and the other amino acid is the C-terminus.

But...odds are we may want a longer polypeptide than two amino acids. How does the chain continue to grow? Once the peptide bond is formed, the mRNA is pulled onward through the ribosome by exactly one codon. This shift allows the first, empty tRNA to drift out via the E ("exit") site. It also exposes a new codon in the A site, so the whole cycle can repeat.

And repeat it does...from a few times up to a mind-boggling $33,$33, comma$000$000 times! The protein titin, which is found in your muscles and is the longest known polypeptide, can have up to $33,$33, comma$000$000 amino acids$^{8,9}$start superscript, 8, comma, 9, end superscript.

Termination :

Polypeptides, like all good things, must eventually come to an end. Translation ends in a process called termination. Termination happens when a stop codon in the mRNA (UAA, UAG, or UGA) enters the A site.

Stop codons are recognized by proteins called release factors, which fit neatly into the P site (though they aren't tRNAs). Release factors mess with the enzyme that normally forms peptide bonds: they make it add a water molecule to the last amino acid of the chain. This reaction separates the chain from the tRNA, and the newly made protein is released.

What next? Luckily, translation "equipment" is very reusable. After the small and large ribosomal subunits separate from the mRNA and from each other, each element can (and usually quickly does) take part in another round of translation.

conclusion :

Our polypeptide now has all its amino acids—does that mean it's ready to do its job in the cell?

Not necessarily. Polypeptides often need some "edits." During and after translation, amino acids may be chemically altered or removed. The new polypeptide will also fold into a distinct 3D structure, and may join with other polypeptides to make a multi-part protein.

Many proteins are good at folding on their own, but some need helpers ("chaperones") to keep them from sticking together incorrectly during the complex process of folding.

Some proteins also contain special amino acid sequences that direct them to certain parts of the cell. These sequences, often found close to the N- or C-terminus, can be thought of as the protein’s “train ticket” to its final destination.