Question

Describe the process of protein synthesis in prokaryotic cells.

Begin with a gene in the DNA and throughly explain all of the steps of transcription (initiation , elongation, termination and one type of termination.

Describe translation(initiation, elongation, termination) that result in the formation of a functional protein. Be specific as to the mechanism of each step.

Answer 1

Process of protein synthesis in prokaryotic cells. Protein synthesis is the synthesis of proteins and occurs in the cytoplasm. In prokaryotes transcription and translation occurs concurrently (translation starts before the completion of transcription).

Transcription: DNA dependent synthesis of any type of RNA molecule with the help of RNA Polymerase is called transcription. Transcription always start at specific site called promoter. Promoter is specific site on DNA where RNA Polymerase binds and initiates transcription. Synthesis of RNA occurs in 5' to 3' direction and continuous, only a strand of DNA act as template and only a small segment of DNA is transcribed at a time generally called gene. Prokaryotic promoters are always present on upstream to transcription start site, are 70 base pair long and contain two consensus sequence of 6 base pair at -10 region and -35 region (-10 region is also known as TATA box). In prokaryotes single type of RNA Polymerase transcribe all types of RNA such as mRNA, rRNA and tRNA. RNA Polymerase is a multisubunit enzyme and consists of five different polypeptides called (holoenzyme). subunit helps in promoter recognition and initiate transcription whereas core enzyme consists of      subunits and catalyze transcription. Prokaryotic transcription completed in three sequential steps:

• Initiation: The factor associated with core enzyme to form holoenzyme, the holoenzyme binds with promoter to form closed binary complex. Unwinding of double strand is done which creates an open complex. Addition of first nucleoside triphosphate to form ternary complex and   factor is released as RNA Polymerase moves.
• Elongation: Once RNA Polymerase successfully synthesize a short stretch of RNA. RNA transcript synthesized by RNA Polymerase continuously dissociates from DNA template as well as transcription proceeds. Only 8 to 10 base pair long RNA-DNA complementary is maintained in transcription bubble region.
• Termination: Transcription termination occurs at the end of gene and is always guided by transcribed RNA molecule because transcription termination signal present in RNA. Termination occurs by two types:

1. Rho dependent termination: Have two common features: A DNA sequence that pause transcription and an ill-defined RNA element called rut site (rut utilization). Rho protein binds with rut site and moves towards its 3'end. When RNA Polymerase encounters terminator sequence it pause and catch by rho and interaction of rho by RNA Polymerase. Using RNA-DNA helicase activity rho unwinds RNA-DNA hybrid and release RNA transcript.

2. Rho independent termination: Have two features: Hair pin loop structure includes GC rich region which prevents the movement of RNA Polymerase. A stretch of adenine residue in template strand at the end of gene.

Translation: Translation completed in four sequential steps:

• Activation of amino acid (tRNA charging): Amino acid activation occurs when a specific amino acid covalentally binds with 3'end of its cognate tRNA. Activation of amino acid is catalysed by an ATP dependent enzyme called aminoacyl tRNA synthetase (AATS).
• Initiation: Completed in three sequential steps: 1) Small subunit of ribosome binds with mRNA and is facilitated by two proteinaceous factors called IF-1 and IF-3. 2) Binding of initiating charged tRNA, binding of charged initiating tRNA to the P site of small subunit of ribosome catalysed by IF-2 in GTP bound state. 3) Binding of large subunit with small subunit to form 70S initiation complex.
• Elongation: Elongation phase of translation includes codon to codon translocation of active ribosomes on mRNA in 5' to 3' direction and forms peptide bond between codon specified amino acid. Completed in three sequential steps: 1) Binding of aminoacyl tRNA (charged tRNA) to the A site of ribosomes and is catalysed by GTP bound elongation protein EF-Tu. 2) Peptide bond formation, after the release of EF-Tu amino group of A site amino acid act as nucleophile on carboxylic group of P site amino acid and makes peptide bond. After peptide bond formation P site tRNA becomes uncharged and A site tRNA generates dipeptidyl tRNA. 3) Translocation of ribosomes, translocation shifts dipeptidyl tRNA from A site to P site and uncharged tRNA from P site to E site and A site occupy next codon thus open for incoming aminoacyl tRNA.
• Termination: Translation termination occurs when A site occupies a stop codon . In this case, A site not entered by an aminoacyl tRNA but by proteinaceous factor called release factor.