Sorting of Proteins.

Protein Sorting

Protein synthesis is initiated on ribosomes in cytosol except for those which are synthesised on the ribosomes of plastids. Most proteins do not have sorting signals & remain permanent resident of cytosol. Many others have specific sorting signals that direct their transport to different organelles.

There are three fundamental ways of protein transport:

1. Active transport through pores.
2. Transmembrane transport by a membrane bound translocator.
3. Vesicular Transport.

All the three mechanisms are guided by sorting signals which are mainly of two types:

1. Signal Peptide : A continuous stretch of 15 – 16 amino acids on the polypeptide at either terminal, which may be cleaved post sorting. (fig.A). Signal peptide direct the protein from cytosol to various orgaelles
2. Signal Patch : A specific 3D arrangement of amino acids on protein surface, once it folds. This portion may not be a linear stretch of amino acids hence signal patch (fig.B) Signal patches identifyenzymes that glycosylates them. Once glycosylated, the sugar residue then direct such proteins from Golgi to other target organelles.

Organelle specific Sorting of Proteins

Peroxisomes

· All peroxisomal proteins are synthesised on cytosolic ribosomes and incorporated post translationally.

· Most proteins have signal sequences at C – terminal while a few have at N – terminal. Signal sequences are part of functional proteins, hence not cleaved.

· Signal sequences are specific 3 amino acid sequence at either terminal.

· Many proteins are imported in a folded state across the peroxisomal membrane. This translocation involves ATP hydrolysis.

· Proteins incorporated in peroxisomal membrane & peroxisomal matrix have different signal sequence.

Mitochondria and Chloroplast

· Plastid proteins that are encoded by nuclear genes & synthesised by cytosolic ribosomes are imported post-translationally, but in an unfolded state. Cytosolic chaperons maintain the unfolded state.

· Signal sequence lies at N-terminal & is always cleaved after transporting into matrix.

· Signal sequence for mitochondria have alternate specific positively charged amino acids at one terminal and hydrophobis at another.

· Signal sequence for chloroplast on the other hand is the protein itself, rich in serine, threonine and other small hydrophobic amino acid but poor in aspartate & glutamate.

· Translocation occurs at a site where outer and inner membranes are close together.

· Protein is first transported into matrix and then redirected to its destination. Hence two or more signal sequences (one directing transport from cytosol to plastid matrix and the rest direct it to its destination in plastid itself.)

Endoplasmic Reticulum (ER)

· Proteins are transported into ER either post-translationally or cotranslationally. Latter requires direct association of cytosolic ribosome to ER achieved by Signal Recognition Particle, SRP.

· While polypeptide is being synthesised, SRP recognises signal sequence, binds to it, then to ribosome and targets the whole to ER at a specific receptor.

· Binding of SRP to ribosome stalls translation temporarily. Once the whole assembly binds at ER, SRP unbinds from polypeptide signal & SRP receptor at ER. This allows resumption of polypeptide synthesyis by ribosome.

· The growing polypeptide is pushed into ER through a translocon channel and subsequently released into lumen of ER by cleaving signal sequence (always) by enzyme signal peptidase.

· In post translational transport, poypeptides that are destined to be permanent resident of ER, the signal sequence consists of specific 4 amino acids at C - terminal, wwhile those which are further directed into Golgi from ER have signal that comprises of 5-10 hydrophobic amino acids at N – terminal.

Details of co-translational sorting into ER.

Proteins that are sorted cotranslationally contain a signal sequence at N -terminal of the growing polypeptide. The main step in such transport is association of ribosome to ER membrane via a Signal Recognition Particle (SRP).

SRP is a G-protein consisting of 6 polpeptides & 7s RNA. Main steps of sorting are:

1. SRP binds to signal sequence of polypeptide and also ribosome, inhibits the translation.

2. It then targets the whole assembly of ribosome, mRNA, polypeptide, and itself to ER. There it binds at a SRP receptor on ER membrane. The ribosome also attaches itself to SRP receptor.

3. Shortly after SRP releases itself from assembly facilitated by GTP hydrolysis. The signal peptide is thus released into translocon. Translocon is made up of 3 membrane proteins – sec61, it is gated channel, 50’A in diameter

4. Release of SRP allows ribosome to resume translation. At this point ribosome completely blocks the translocon. The growing polypeptide is directly transferred into lumen of ER through translocon.

5. Signal Peptidase is bound to an internal site of translocon and serves the purpose of cleaving signal sequence.

...Continue reading

Gene regulation - An Introduction

Gene expression can be controlled at any of several stages, which we divide broadly into transcription, processing, and translation:

• Transcription often is controlled at the stage of initiation. Transcription is not usually controlled at elongation, but may be controlled at termination to determine whether RNA polymerase is allowed to proceed past a terminator to the gene(s) beyond.

• In eukaryotic cells, processing of the RNA product may be regulated at the stages of modification, splicing, transport, or stability. In bacteria, an mRNA is in principle available for translation as soon as (or even while) it is being synthesized, and these stages of control are not available.
• Translation may be regulated, usually at the stages of initiation and termination (like transcription). Regulation of initiation is formally analogous to the regulation of transcription: the circuitry can be drawn in similar terms for regulating initiation of transcription on DNA or initiation of translation on RNA.

The basic concept for how transcription is controlled in bacteria was provided by the classic formulation of the model for control of gene expression by Jacob and Monod in 1961 (Jacob and Monod, 1961). They distinguished between two types of sequences in DNA: sequences that code for trans-acting products; and cis-acting sequences that function exclusively within the DNA. Gene activity is regulated by the specific interactions of the trans-acting products (usually proteins) with the cis-acting sequences (usually sites in DNA). In more formal terms:

• A gene is a sequence of DNA that codes for a diffusible product. This product may be protein (as in the case of the majority of genes) or may be RNA (as in the case of genes that code for tRNA and rRNA). The crucial feature is that the product diffuses away from its site of synthesis to act elsewhere. Any gene product that is free to diffuse to find its target is described as trans-acting.
• The description cis-acting applies to any sequence of DNA that is not converted into any other form, but that functions exclusively as a DNA sequence in situ, affecting only the DNA to which it is physically linked. (In some cases, a cis-acting sequence functions in an RNA rather than in a DNA molecule.

To help distinguish between the components of regulatory circuits and the genes that they regulate, we sometimes use the terms structural gene and regulator gene. A structural gene is simply any gene that codes for a protein (or RNA) product. Structural genes represent an enormous variety of protein structures and functions, including structural proteins, enzymes with catalytic activities, and regulatory proteins. A regulator gene simply describes a gene that codes for a protein (or an RNA) involved in regulating the expression of other genes.

..

Figure 10.1  
A regulator gene codes for a protein that acts at a target site on DNA.

Download

The simplest form of the regulatory model is illustrated in Figure above: a regulator gene codes for a protein that controls transcription by binding to particular site(s) on DNA. This interaction can regulate a target gene in either a positive manner (the interaction turns the gene on) or in a negative manner (the interaction turns the gene off). The sites on DNA are usually (but not exclusively) located just upstream of the target gene.

...Continue reading

Polymerase chain Reaction

...Continue reading

Plant Transgenesis

...Continue reading

Sitemap

Enter your email address:


Delivered by FeedBurner

...Continue reading