Molecular BiologyTelomerasesTranscription Introduction

Dr. Aga Syed SameerCSIR Lecturer (Demonstrator)Department of Biochemistry,Medical College,Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190018. India.

Replication - Termination• In E. coli the two replication forks meet almost 180o

opposite to oriC, around this region there are multiple copies of a 20bp sequence called as Ter which act as a trap for the replication fork

• Ter sequence are actually binding site for a protein called Tus(terminus utilization substance)

• Tus-Ter complex arrests the replication fork from only one direction (which one comes first) and hence prevents over replication by the polymerase

• The final few hundred base pairs of DNA between the two arrested forks are then replicated producing two DNA circles which are topologically interlinked called as catenates

• Separation of catenates is then done by topoisomerase IV thus completing replication

Telomerases• In eukaryotes the ends of the linear chromosomes are

replicated in a special manner by the help of enzymes called as telomerases, without the action of which a chromosome would be shortened at both ends by 50-100 ntwith every cycle of DNA replication and cell division

• Telomerase is actually a cellular reverse transcriptase in its function as it contains a 150nt RNA molecule and a protein component having a 5’ →3’polymerase activity

• RNA in it contains about 1.5 copies of the appropriate CyAxtelomere repeat

Telomerases• As telomere binds to the ends of the chromosome, it uses 3 -׳ OH

group of the leading strand of the double stranded daughter DNA molecule as the site for the addition of new bases and its own RNA molecule as the template to direct the synthesis of the telomere repeat

• After having synthesized one copy of the repeat , the enzyme repositions itself to resume the extension of the telomere

• In the second phase complementary CyAx strand is synthesisedby the DNA polymerase using this telomere repeat as its template as it would have during the normal lagging strand synthesis

• The nick at last is sealed by the DNA ligase

Molecular BiologyTranscriptionMachinery

Transcription

• This process involves the synthesis of the single stranded RNA from the double stranded DNA molecule

• However out of the two strands of the DNA molecule, only one stand acts as the parent-sequence provider for the RNA synthesis

• Called as the template or antisense strand

• The other is called as the non-template or sense strand

• It is also called as coding strand as its sequence is identical to RNA that is being transcribed from the template strand

Transcription

Transcription

• The synthesis of RNA on the sequence of the template DNA occurs in the 5’→3’ direction similar to DNA synthesis

• The process is the enzymatic synthesis taking place with the help of DNA-dependent RNA polymerases

• In addition to a DNA template, all four ribonucleoside 5-triphosphates (ATP, GTP, UTP, andCTP) as precursors of the nucleotide units of RNA, as well as Mg ions and Zn ions for stabilisation

Transcription Bubble

• The DNA duplex must unwind over a short distance, forminga “transcription bubble” to allow RNA synthesis to takeplace on template DNA strand

• During transcription, the E. coli RNA polymerase generallykeeps about ~18 bp unwound

• The 8-9 bp RNA-DNA hybrid occurs in this unwound region

• Elongation of a transcript by E. coli RNA polymeraseproceeds at a rate of 50 to 90 nucleotides/s

Transcription Machinery

• RNA polymerases in E. coli

• In E. coli all the genes are transcribed by a single large RNA polymerase

• The enzyme consists of core enzyme of five subunits:

• {α2ββ΄ω; of molecular weight of ~40 KD}and

• a sixth subunit called as sigma factor σ

• Thus a complete holoenzyme is {α2ββ΄ωσ}

• α subunit is the product of rpoA gene and is required for the proper promoter recognition and binding of the polymerase

• β subunit is the product of rpoB gene and is required for the transcription initiation and elongation. It is inhibited by rifampicin and streptolydigin

• β ׳subunit is the product of rpoC gene, it plays role in the catalytic function of the polymerase. It binds two zinc ions which facilitate in its function

Transcription Machinery

• RNA polymerases in E. coli

• The complete holoenzyme is required for the transcription initiation as σ factor has a critical role in promoter recognition

• However, is not required for transcription elongation and is released from the transcription complex soon after the transcription initiation

• The σ factor decreases the affinity of the core enzyme for non-specific DNA site by a factor of 104 & increases affinity for the promoter

• RNA polymerases lack proof reading 3'→ 5' exonucleaseactivity and hence one error is made for every 104 to 105

ribonucleotides incorporated into the RNA during transcription

Transcription Machinery

• RNA polymerases in Eukaryotes

• The transcriptional machinery of the eukaryotes is much more complex than that of the prokaryotes

• Although the mechanism remains the same but eukaryotes contain three different RNA polymerase complexes for the transcription of different types of eukaryotic genes

• These were identified by chromatographic purification of the enzymes

• Each RNA polymerase has a different sensivity to the fungal toxin α-amanitin and this is used to distinguish their activities

Transcription Machinery

• RNA polymerase I: It is located in nucleoli and is insensitive to α-amanitin. It transcribes polycistronic rRNA called as preribosomal RNA which contains the precursor for the 5.8S, 18S and 28S rRNAs

• RNA polymerase II: It is located in nucleoplasm and is very sensitive to α-amanitin. It is central polymerase and transcribes all protein-coding genes i.e. synthesizes mRNA

• RNA polymerase III: It is located in nucleoplasm and is moderately sensitive to α-amanitin. It transcribes the genes for tRNA, 5S rRNA, U6 snRNA and certain other small RNAs

• All three polymerases are large enzymes containing 12 or more subunits. The genes encoding the two largest subunits of each RNA polymerase have homology to each other. All of the three eukaryotic polymerases contain subunits which have homology to subunits within the E. coli core RNA polymerase

Questions?