Question

Biochem question

COMPLETE THE FOLLOWING QUESTIONS FOR TRANSCRIPTION IN PROKARYOTES: Next four (4) questions Enzyme that catalyze RNA synthesis: RNA polymerase Name of the subunit that recognizes the promoter site: Your answer Name the two signals for the synthesis termination: Your answer Regulation mechanism:

Answer 1

The transcription in prokaryotes is relatively simple compared to that of eukaryotes. The enzyme RNA POLYMERASE (RNAP) catalyzes the synthesis of all types of RNA in prokaryotes. RNA polymerase contains 5 polypeptide subunits: 2 Alpha, 2 beta and 1 sigma. The sigma factor is responsible for the recognition of the promoter element, whereas the rest of the enzyme catalyzes the RNA synthesis. Transcription occurs in three stages: 1) Initiation 2) Elongation 3) Termination.

INITIATION

The process of RNA synthesis in bacteria begins with the binding of RNA polymerase molecule on the DNA. RNA polymerase recognises the transcription start site on the template strand of DNA with the help of certain specific regions on the DNA known as PROMOTER ELEMENTS (PE). The sigma factor plays this role. There are two PE regions in bacteria.

1. Pribnow box (TATA BOX) : this component of PE is a sequence of 6 nucleotide bases (TATAAT) that occurs 10 base pairs upstream to the transcription initiation point.
2. The '-35- sequence: the second component of promoter element with a consensus sequence of eight nucleotide bases (TGTTGACA) occurs 35 base pairs upstream to the transcription initiation site (TIS).

Soon after the binding of RNA polymerase to the promoter element, the first nucleotide (always a purine) is inserted at the 5' end of the nascent RNA moleculeand the RNA polymerase moves next to the second base in the template. Phosphodiester bond forms with the second nucleotide base and the transcription is initiated. At this stage, the sigma factor of RNA polymerase dissociates from the rest of the enzyme.

ELONGATION

The elongation of the RNA molecule occurs from the 5' end to 3' end, antiparallel to the template strand. RNA polymerase assembles ribonucleotides in a complementary sequence to the template strand.

Nucleotide base sequence in the new RNA is identical to that of the coding strand of DNA, except that it has U in place of T in the DNA. Unlike DNA polymerase during replication, RNA polymerase does not possess proofreading ability.

TERMINATION

Transcription ends by certain termination signals.

1. Rho factor: It is a protein that binds either to the growing RNA or template strand of DNA and dissociates RNA polymerase from DNA, thus terminating transcription. The released RNA polymerase assembles with sigma factor and is recycled.
2. Palindromes: These are words that read alike backwards and forwards, for example: rotor, Malayalam, etc. There occur pallindrome like base sequences at the end portions of genes. Due to these sequences, the newly synthesized RNA folds on itself to form a hairpin like structure. This terminates RNA synthesis.

Enzyme catalysing RNA synthesis: RNA polymerase

Subunit recognising Promoter element: sigma factor.

Two signals for termination: Rho factor and Palindromes

Regulation mechanism:

It is done with help of operon. Operon is a unit consisting of regulatory elements and Protein encoding genes which act in a well orchestrated manner. Operon is considered a coordinated unit of gene expression. It is done by Lac operon.

most bacteria such as E coli utilise glucose as a fuel source. But when glucose is unavailable it uses lactose as the alternative fuel source. Three enzymes are said to be involved in the lactose metabolism: Beta-galactosidase, Permease, transacetylase. Beta galactosidase hydrolyzes lactose into galactose and glucose which are then further metabolism. Permease is required for the transport of lactose into the cell. Transacetylase function is not yet known.

Structure: the Lac operon is composed of a regulatory gene'I' (I for inhibition), an operator gene (O) and a set of three structural genes (Z,Y,A) . It also contains a promoter site (P) adjacent to the operator gene which directs the RNA polymerase to correct the transcription start site. Z, Y and A genes are transcribed into a single large mRNA which encodes for three enzymes. This is called polycistronic mRNA.

I is expressed to form a Repressor protein which has high affinity for operator gene O. The binding of lac repressor to the operator gene interferes with the function of promoter site (which directs RNAP to the correct transcription start site). This prevents the transcription of three structural genes. In this way the Lac operon is frozen in a state of repression.

When E coli cells are exposed to lactose, the repressor molecules which have a higher affinity for lactose bind to it quickly. The lactose molecules induce some conformational changes in the lac repressor. This reduces the repressor's affinity for the operator gene and is delinked from it. Once the operator gene is free from the repressor, the promoter element directs the RNA polymerase to bind to the transcription start site and the structural genes are transcribed into polycistronic mRNA and then translated into three enzymes. This is called derepression.

E-Coli prefers glucose over lactose as a fuel source. It utilizes lactose only when Glucose levels are depleted. This indicates that lac operon is in a repressed state. A regulatory protein called CAP( catabolic gene activator protein) bound to cyclic AMP acts as a positive regulator of the Lac operon. When glucose levels are high in the cells, glucose inactivates the enzyme adenylyl cyclase which is responsible for the synthesis of cyclic AMP from ATP. The lowered levels of cyclic amp interferes with the activity of CAP which in turn hampers the transcription of structural genes in the lac operon.