Question

41. What are the main differences of the gene to protein axis between eukaryotic and prokaryotic systems ? Please write/explain the following terms, genetic code, reading frame, six amino acids with their abbreviation, start and stop codons. 45. Please explain the amino acid activation process for tRNA, and aminoacyl-tRNA linkage with mediated proteins in detail. 61. Please explain the mechanism of eukaryotic translation initiation in detail. 65. Which technique do you know that you can separate a DNA fragment according to its protein binding capacity ? Please explain how you can do it ? 81. What is the difference of Selenocysteine, when we compared to other amino acids. Please explain in detail.

45. Please explain the amino acid activation process for tRNA, and aminoacyl-tRNA linkage with mediated proteins in detail.

61. Please explain the mechanism of eukaryotic translation initiation in detail.

65. Which technique do you know that you can separate a DNA fragment according to its protein binding capacity ? Please explain how you can do it ?

81. What is the difference of Selenocysteine, when we compared to other amino acids. Please explain in detail.      

Answer 1

41.

PROKARYOTES	EUKARYOTES
Prokaryotic gene expression is primarily controlled at the level of transcription	Eukaryotic gene expression is controlled at the levels of epigenetics, transcription, post-transcription, translation, and post-translation.
Prokaryotic gene expression (both transcription and translation) occurs within the cytoplasm of a cell due to the lack of a defined nucleus; thus, the DNA is freely located within the cytoplasm.	Eukaryotic gene expression occurs in both the nucleus (transcription) and cytoplasm (translation).
The regulation of transcription is the primary method to control what type of protein and how much of each protein is expressed in a prokaryotic cell.	Eukaryotic gene expression is regulated during transcription and RNA processing, which take place in the nucleus, and during protein translation, which takes place in the cytoplasm.
Prokaryotes regulate gene expression by controlling the amount of transcription	Eukaryotic control is much more complex.

• GENETIC CODE:The genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells.Those genes that code for proteins are composed of tri-nucleotide units called codons, each coding for a single amino acid.
• READING FRAME:One of the three possible ways of reading a nucleotide sequence. As the genetic code is read in nonoverlapping triplets (codons) there are three possible ways of translating a sequence of nucleotides into a protein, each with a different starting point. For example: given the nucleotide sequence: AGCAGCAGC, the three reading frames are: AGC AGC AGC, GCA GCA, CAG CAG.
• AMINO ACIDS:

Amino acid	Abbrevation
Alanine	Ala
Arginine	Arg
Glutamine	Glu
Aspartate	Asp
Methionine	Met
Phenylalanine	Phe

• Start and Stop codons:The start codon marks the site at which translation into protein sequence begins, and the stop codon marks the site at which translation ends.
• Examples:UGA, UAA, and UAG are stop codons. AUG is the codon for methionine, and is also the start codon.

45.

• Each tRNA molecule binds with a specific amino acid in the cytoplasm in a reaction catalysed by a tRNA-activating enzyme

• Each amino acid is recognised by a specific enzyme (the enzyme may recognise multiple tRNA molecules due to degeneracy)

• The binding of an amino acid to the tRNA acceptor stem occurs as a result of a two-step process:

• The enzyme binds ATP to the amino acid to form an amino acid–AMP complex linked by a high energy bond (PP released)
• The amino acid is then coupled to tRNA and the AMP is released – the tRNA molecule is now “charged” and ready for use.

• The function of the ATP (phosphorylation) is to create a high energy bond that is transferred to the tRNA molecule

• This stored energy will provide the majority of the energy required for peptide bond formation during translation.
• The aminoacyl-tRNA synthetases catalyze the covalent linkage of amino acids to their cognate set of tRNA isoacceptors. In order to ensure the fidelity during protein synthesis some synthetases also perform amino acid editing function.
• These enzymes have structurally and functionally evolved to host diverse alternate activities unrelated to their primary roles in protein synthesis.
• In higher eukaryotes several synthetases assemble to form macromolecular complexes via protein–protein interactions. Also various human disease conditions have been associated with these enzymes due to their functional versatility.

61.

• Eukaryotic translation initiation and ribosomal subunit recycling are depicted as a nine-stage process. In stage 1, ribosome recycling occurs to yield separated 40S and 60S ribosomal subunits.
• In stage 2, eukaryotic initiation factor 2 (eIF2), GTP, and an initiator methionine tRNA (Met-tRNAMeti) form a ternary complex called eIF2–GTP–Met-tRNAMeti.
• In stage 3, the 43S preinitiation complex forms. This complex includes a 40S subunit, eIF1, eIF1A, eIF3, eIF2–GTP–Met-tRNAMeti and probably eIF5.
• In stage 4, mRNA activation occurs, during which the mRNA cap-proximal region is unwound in an ATP-dependent manner by eIF4F with eIF4B. The mRNA loops into a circular configuration.
• In stage 5, the 43S preinitiation complex attaches to the unwound mRNA region.
• In stage 6, the 43S complex scans the 5′ UTR in a 5′ to 3′ direction.
• In stage 7, the initiation codon is recognized, and the 48S initiation complex forms, which switches the scanning complex to a 'closed' conformation. This leads to the displacement of eIF1 to allow eIF5-mediated hydrolysis of eIF2-bound GTP and inorganic phosphate (Pi) release.
• In stage 8, the 60S subunit joins the 48S complex, and there is concomitant displacement of GDP-bound eIF2 and other factors (eIF1, eIF3, eIF4B, eIF4F, and eIF5) mediated by eIF5B.
• In stage 9, hydrolysis of eIF5B-bound GTP occurs, and eIF1A and GDP-bound eIF5B are released from the assembled elongation-competent 80S ribosomes. Translation is a cyclical process. Following elongation, termination occurs, followed by recycling (stage 1), which generates separated ribosomal subunits, and the process begins again.
• 

65.

• Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge.
• Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another.
• All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of DNA fragments separates them based on size only.
• Using electrophoresis, we can see how many different DNA fragments are present in a sample and how large they are relative to one another.

81.

• Selenocysteine is an unusual amino acid of proteins, the selenium analogue of cysteine, in which a selenium atom replaces sulphur. Involved in the catalytic mechanism of seleno enzymes such as formate dehydrogenase of E. coli and mammalian glutathione peroxidase.
• Selenocysteine, the 21st amino acid, has been found in 25 human selenoproteins and selenoenzymes important for fundamental cellular processes ranging from selenium homeostasis maintenance to the regulation of the overall metabolic rate.
• Selenocysteine is located in the active sites of enzymes that participate in oxidation–reduction reactions.
• Their deregulation is being associated with neurodegenerative and other diseases, for which the underlying mechanisms have remained unclear. However, using novel transgenic mouse models they found that reducing the expression of selenoproteins in the mammalian brain impairs brain development and healthy functioning, consequently causing a broad spectrum of disorders, including epilepsy and neurodegeneration.