RNA SYNTHESIS

Mukund Modak, Ph.D.

Lecture 32 Modak

U1-U6 <100 nt 1 SplicingRNAs

PROKARYOTIC RNA POLYMERASE

Single enzyme with 5 subunits

’’ : Holoenzyme (Sigma subunit finds start point)’’Core enzyme (elongation of RNA chain)

4 rNTPS RNA + PPiDNA template and

RNA polymerase

3’ 5’

5’ 3’

5’ 3’

Figure 30.8 Local unwinding of DNA caused by RNA polymerase

ENZYMATIC SYNTHESIS OF RNA

Differences/similarities with DNA polymerases

i. Substrate

ii. Template conservation (Sense strand = mRNA)

iii. Primer need

iv. Proof reading

Template recognition and copying by base pairing

direction of synthesis : 5’ - 3’

Initiation with A or G (Sigma factor also needed)

*Termination : U sequence + hairpin + factor

* Prokaryotic system (ATP dependent)

Figure 30.7 Structure of the prokaryotic promoter region

EUOKARYOTES PROMOTERS

-110 -60 -25CAAT GCGC TATA (box)

EUKARYOTIC RNA POLYMERASES

RNA polymerase I : (rRNA) in NucleoliRNA polymerase II: (mRNA)RNA polymerase III: (tRNA)

Nucleoplasmic

Figure 30.13 A. Eukaryotic general transcription factors bound to the promoter. CTF, SP1, and TFIID are general transcription factors. B. Enhancer stimulation of RNA polymerase II

Figure 30.17

Posttranscriptional modification of mRNA showing the 7-methylguanosine cap and poly-A tail

RNA Synthesis

• miRNAs (Micro-RNAs): typically 21 to 25 base long and is produced from stem loop structured precursors RNAs.

• Involved in destruction of specific gene transcripts and suppression of translation of specific mRNAs.

• SiRNA or RNAi: small interfering RNAs similar to miRNAS, produced by RNAase III enzyme.

• Functions in  Silencing of mRNA

• 7SL RNA: RNA required in transport (to Golgi) of secretory proteins. 

• SN-RNA: (small Nuclear RNAs) form RNPs required in processing of mRNA.

• Oligo A: (20-50 nucleotide long) stretch of A nucleotides linked from 2’ to 5’. Aids in interferon action.

• tRNAs : Amino acid specific tRNAs required in protein synthesis.

Small RNAs and their functions (all require to form protein complexes)

TRANSFER & RIBOSOMAL RNA SYNTHESIS

Synthesized as long transcripts

16 S tRNA 23S 5S

At arrows - spacer sequences

Cleaves at spacer (marked as arrows) sequences with specific

RNase

CCA ends are added to all. tRNAs

Modifications of some bases, e. g. methylation in both tRNA

and rRNA

INHIBITORS/DRUGS OF RNA SYNTHESIS

1. Enzyme specific

Rifamycin for E. coli enzyme

-amanitin for pol II

2. DNA intercalating agents

Actinomycin D

Quinacrine

3. Substrate analogs

Ara - C

Ara - A