RNA Synthesis(Transcription)MS. NEHAAsst. Prof. (Zoology)

Central Dogma?• Central dogma of molecular biology proposes a

unidirectional or one way flow of information from DNA to RNA (transcription) and from RNA to protein (translation).

• The concept was given by Watson and Crick.

• First step is transcription (synthesis of RNA from DNA), but in case of reverse transcription DNA is synthesizes from RNA in retrovirus. That concept is given by Temin and Baltimore in Rous sarcoma virus, also known as teminism and enzyme catalyze this reaction is reverse transcriptase or RNA dependent DNA polymerase.

• In second step, messenger RNA (mRNA) moves the information from the DNA to the ribosomes to direct the production of protein.  Translation represents a change in the language from the nucleotide letters in RNA to the amino acid letters in protein

Transcription

• RNA synthesis (transcription) is a transfer of the information from the DNA where it is stored into RNA which can be transported and interpreted

• Three Major Classes of RNA These are synthesized by transcription of the appropriate genes and are involved in protein synthesis.   1) mRNA- carries the message from the DNA to the ribosome. 2) rRNA- are major structural components of the protein-synthesizing ribosome. 3) tRNA- act as adaptor molecules in aligning the amino acids according to the sequence present in the mRNA.

Transcription in Prokaryotes • Transcription is catalyzed by RNA polymerase which makes RNA

using DNA as a template.

• The strand of DNA which is transcribed is the template/sense/non-coding strand in 3’-5’ direction.

• The other strand is called non sense/non template/coding strand which is in 5’-3’ direction.  

• Prokaryotes have only one RNA polymerase which synthesis all the three types of RNA’s.

RNA polymerase moves along the template strand of the DNA in the 3’ to 5’ direction, and the RNA molecule grows in the 5’ to 3’ direction.

RNA polymerase consists of five polypeptide chains-β,β’,2 α & σ.

The σ subunit recognizes promoter on DNA template.

β subunit helps in formation of phosphodiester bond

β’ subunit helps in binding with template DNA

Fig. RNA polymerase

Stages involved..

Binding - DNA promoter region  is a stretch of about 40 bp adjacent to and including the transcription start point. Promoter have a start point (designated +1 and usually an A), the six-nucleotide -10 sequence, and the six-nucleotide -35 sequence, located approx. 10 nucleotides and 35 nucleotides upstream from the start point.

• RNA polymerase binds at promoter by σ subunit.

Initiation • RNA synthesis starts from start point• Ribonucleotide triphosphates(rNTPs) acts as substrate and

hydrogen bonded to complementary bases of template strand at start point

• RNA polymerase form phosphodiester bond between 3’-OH end of 1st rNTP and 5’-P end of next rNTP & PPi releases

• After adding upto 9 rNTPs, σ subunit detaches from RNA polymerase enzyme

Fig. Initiation of transcription

Elongation • Successive rNTPs are added to 3’-OH end of growing RNA

chain about 18 base pairs of DNA are unwound

• The most recently synthesized RNA is still hydrogen-bonded to the DNA, forming a short RNA-DNA hybrid(about 12 bp long)

• This unwinding and rewinding causes by RNA polymerase but supercoiling is reduced by topoisomerase

Fig. Elongation of transcription

Fig. Transcription process in prokaryotes

Termination Rho independent

• The newly formed RNA has GC rich sequence followed by poly U sequence

• GC rich sequence form hairpin loop which pulls RNA chain and weak bond between A=U broken & RNA released

Rho dependent • Rho factors/proteins causes

disruption of RNA-DNA hybrid• Rho protein is a specialized

helicase which recognizes 50-90 bp upstream of terminator in mRNA sequence

• Rho protein releases newly formed RNA

Transcription in EukaryotesThree types of RNA polymerases:-

Name Location Productα-Amanitin sensitivity

RNA polymerase I (Pol I, Pol A)

Nucleolus larger ribosomal RNA (rRNA) (28s,18s and 5.8s)

Resistant

RNA Polymerase II (Pol II, Pol B)

nucleusmessenger RNA ,most small nuclear RNAs ,small interfering RNA and micro RNA

Very sensitive

RNA Polymerase III (Pol III, Pol C)

nucleus (and possibly the nucleolus-nucleoplasm interface)

transfer RNA ,other small RNAs (including the small 5S ribosomal RNA (5s rRNA) ,snRNA U6, signal recognition particle RNA (SRP RNA) and other stable short RNAs

Moderately sensitive

Mitochondria Mitochondria Mitochondrial RNA’s Resistant

Chloroplast Chloroplast Chloroplast RNA’s Resistant

-180 -107 -45 +1 +20Transcription

5’ 3’

Fig. Promoter of RNA polymerase I

Fig. Promoter of RNA polymerase II

Fig. Promoter of RNA polymerase III

Initiation • Transcription factors and

the polymerase undergo binding to initiate transcription

1) TFIID binds to the TATA box followed by 2) The binding of TFIIA and TFIIB. 3) The resulting complex is now bound by the polymerase, to which TFIIF has already attached. 4) The initiation complex is completed by the addition of TFIIE, TFIIJ, and TFIIH. 5) Activation step requiring ATP-dependent phosphorylation of the RNA polymerase molecule which initiates transcription

Elongation

• Elongation in eukaryotes is same as in prokaryotes

Termination RNA polymerase I – requires protein factors which recognize 18 nucleotides terminal sequence in newly formed RNA and then termination occurs

RNA polymerase II – recognizes poly U sequence on template DNA but hairpin loop is not formed

RNA polymerase III – newly formed mRNA cleaved at 10-35 nucleotides downstream from AAUAAA sequence in growing mRNA. At cleaved site in mRNA poly A tail is formed

Fig. Termination in eukaryotes