transcriptiontranslation Reverse transcription " The Central Dogma of molecular biology" replication

Chapter 10 Transcription (RNA Biosynthesis)

RNA DNA products ： mRNA tRNA rRNA Transcription*: RNA biosynthesis from a DNA template is called transcription. transcription

Enzymes and Proteins involved in transcription ： substrates : NTP ( ATP, UTP, GTP, CTP ) template: DNA enzyme : RNA polymerase the other Protein factors

Chemical reaction-- polymerization reaction: RNA polymerase catalyze formation of Phosphodiester bonds and release pyrophosphate (ppi) RNA precursor RNA polymerase

RNA biosynthesis is similar to DNA biosynthesis*:  Template- DNA  Enzyme—dependent on DNA  Chemical reaction--the formation of Phosphodiester bonds  Direction of synthesis--- 5’ 3’  obey the ruler of base pai red

RNA biosynthesis includes three stages:  Initiation: RNA polymerase binds to the promoter of DNA, and then a transcription “bubble” is formed.  Elongation: the polymerase catalyzes formation of 3’5’-phosphodiester bonds in 5’  3’ direction, using NTP as building units.  Termination: when the polymerase reaches a termination sequence on DNA, the reaction stops and the newly synthesized RNA is released.

Formation of a transcription bubble

1. RNA biosynthesis in prokaryotes

RNA polymerase in E. coli : consists of five subunits,  2  ’ , which is called “holoenzyme”. The  subunit functions as a starting factor that can recognize and bind to the promoter site. The rest of the enzyme,  2  ’ , is known as “core enzyme”, responsible for elongation of the RNA sequence.

RNA-pol 1)Important terms in RNA biosynthesis. A)Operon*: a coordinated unit of gene expression, which usually contains a regulator gene and a set of structural genes. B) Promoter site*: a region of DNA templates that specifically binds RNA polymerase and determines where transcription begins. regulator genestructural genes Promoter site

 The –10 sequence: refers to the consensus TATAAT, and is known as “Pribnow box”.  The –35 sequence: refers to the consensus TTGACA, which is recognized by the  subunit of RNA polymerase, recognition site

consensus sequences T T G A C A A A C T G T -35 (Pribnow box) T A T A A T Pu A T A T T A Py recognition site region the site of transcription (the start site)

C) Sense and antisense strand: The antisense (-) strand refers to the DNA strand that is used as template to synthesize mRNA. The sense (+) strand of a DNA double helix is the non-template strand that has the same sequence as that of the RNA transcript except for T in place of U. Antisense (-) strand = template strand Sense (+) strand = coding strand

coding strand structural gene template strand antisense strand sense strand Sense and antisense strand:

3) Process of RNA biosynthesis: The process is similar to DNA synthesis but no primer is needed and T is replaced by U. A)Initiation: σ factor recognizes the initiation site （ -35 region ）, the holoenzyme of RNA-pol bind to duplex DNA and move along the double helix towards –10 region. the holoenzyme of RNA-pol arrived on –10 region ， and bind to –10 region ， DNA is partially unwound and was opened bp length.

Then incoming 2 neighbour nucleotides which base pairs are complementary with DNA template, RNA polymerase catalyzed the first polymerization reaction. –  5’ -pppGpN – OH + ppi RNApol(α 2 ββ ˊ σ)-DNA-pppGpN-OH3’  pppGNTPpppGpN - OH ppi initiation complex: 5’-pppG - OH + NTP

B) Elongation: after the first phosphodiester bond has been formed, the  subunit is released. The core enzyme moves in a 5’  3’ direction on the DNA strand while it is catalyzing elongation of the RNA transcript.

RNA-pol ( core enzyme) ···· DNA ···· RNA tanscription complex:

C) Termination: when the core enzyme reaches a termination sequence, the region near the 3’end of RNA forms a hairpin structure by self base-pairing. The transcription stops, the core enzyme and the newly synthesized RNA are released. For those DNA templates that lack the sequence to produce a hairpin structure of the RNA transcript, a protein factor called “  ” recognizes the termination site, stops transcription, and causes release of the newly synthesized RNA.

A hairpin structure at the 3’end of RNA

Subunits of RNA polymerase in E. coli Subunit Size (AA) Function  329 required for assembly of the enzyme; interacts with some regulatory proteins; involved in catalysis  1342 involved in catalysis: chain initiation and elongation  ' 1407 binds to the DNA template  613 directs the enzyme to the promoter  91 required to restore denatured RNA polymerase in vitro to its fully functional form

4) Post-transcriptional modification: The newly synthesized precursors of rRNA and tRNA in bacteria undergo a series of process. A) Processing of rRNA: the 16S, 23S, and 5S rRNAs in prokaryotes are produced by cleavage of a rRNA precursor, catalyzed by ribonuclease III. Additional processes include methylation of bases and sugar moieties of some nucleotides.

Processing of rRNAs

B) Processing of tRNA:  The removal of the 5’ end of tRNA precursors is catalyzed by RNase P. RNase P is a ribozyme consisting of RNA that possesses enzyme activity.  Other processes include the addition of nucleotides (CCA) to the 3’-end of tRNA, and formation of some unusual residues such as pseudo-U, I, T, methyl-G, and DHU, etc.

Modification of some residues in tRNAs

4) Inhibition of transcription:  Rifampicin: an antibiotic that specifically inhibits the initiation of transcription by blocking the formation of the first several phosphodiester bonds in RNA biosynthesis.  Streptolydigin: binds to bacterial RNA polymerase and inhibits elongation of RNA chain.  Actinomycin D: binds to DNA and prevents transcription (at low concentrations it doesn't affect DNA replication)

2. RNA biosynthesis in eukaryotes 1)RNA polymerases in eukaryotes: three enzymes, each of which contains 12 or more subunits. Polymerase locationRNAs transcribed Pol I nucleolus28S, 18S, 5.8S rRNA Pol II nucleoplasmpre-mRNA, snRNA Pol III nucleoplasmtRNA, 5S rRNA, U6 snRNA, 7S RNA

2) Process of eukaryotic RNA synthesis A) Initiation: similar to Pribnow box, a start site consensus (called TATA box) at –25 is required for the recognition by RNA polymerase in eukaryotes.

 Pol II requires several transcription factors to start transcription: TFII-A: to stabilize the TFIID-TATA box complex; TFII-B: to link Pol II to the initiation complex; TFII-D: to recognize and bind to the TATA box; TFII-E: to interact with Pol II and TFII-B; TFII-F: to form Pol II-TFIIF complex. It also has DNA helicase activity; TFII-H, -J: to form the initiation complex.

B) Elongation : after the initiation complex has formed, the RNA polymerase catalyzes transcription in a 5’  3’direction, using the (-) DNA strand as template.  Soon after the 5’end of the extending RNA chain appears from the polymerase complex, a cap structure is added at the end.

Cap structure of mRNA 7-methylguanylate

C) Termination: Two mechanisms may cause termination of RNA transcription:  A hairpin structure formed at the 3’end of the nascent RNA causes stop of transcription, as is seen in the prokaryotic RNA synthesis.  A stop signal sequence, AAUAAA, near the 3’end results in the recognition and binding by a specific endonuclease, which cleaves the nascent RNA chain and stops transcription. The newly synthesized mRNA precursor is then added a poly A tail by poly A polymerase.

Cleavage and polyadenylation of a mRNA precursor

3) Processing of eukaryotic RNA precursors: A)Gene organization: protein-coding genes in eukaryotic DNA are organized in a discontinuous fashion. The protein-coding sections are called “exons”, which are interrupted by noncoding sections called “introns”.

B) RNA splicing: a process in which introns of a pre-mRNA are removed to produce a functional mRNA.

C) Steps in RNA splicing: usually the exon- intron boundaries are marked by specific sequences. The intron starts with GU and ends with AG. Intron

I.Formation of a lariat intermediate: the phosphodiester bond of the 5’ splice site is attacked by the 2’-OH of the residue A in the branch point, forming a 2’5’bond and releasing the exon 1 with a new 3’- OH end. II.Connection of exons: The new 3’-OH end attacks the phosphodiester bond at the 3’splice site causing the two exons to join and releasing the intron.

 RNA splicing requires the small nuclear ribonucleoprotein particles (snRNP), each of which consists of a small nuclear RNA and several proteins. They are named U1, U2, U3….  snRNPs bind to the pre-mRNA to form a complex, called spliceosome, which brings the two neighbored exons together for splicing.

Spliceosome

Lariat intermediate

4) Alternative processing: A)Alternative polyadenylation sites: this will cause different splice-sites and produce different mRNAs with varied lifetimes.

B) Alternative splicing: will cause different combinations of exons from a primary transcript of a single gene. This may be resulted from regulatory proteins that control the use of certain splice-sites.

5) RNA editing: refers to the reactions that can change the nucleotide sequence of an mRNA molecule by non-splicing mechanisms. The change may include: nucleotide(s) change, deletion, and insertion. e.g. the mRNA for apolipoprotein B in the liver is translated to apolipoprotein B100, while in the small intestine the mRNA is changed to yield a new termination codon (UAA), resulting in a much shorter protein, apolipoprotein B48.

ApoB100 ApoB48 Edited mRNA

3. Reverse transcription and RNA replication 1)Reverse transcription: biosynthesis of DNA using RNA as a template.  It is important for some viral infections. These viruses are called retroviruses, such as some tumor viruses and HIV.  Reverse transcription is also a powerful tool in molecular biological techniques or genetic engineering, such as RT-PCR.

2)RNA replication:  RNA replication occurs in some viruses. These viruses encode RNA-directed RNA polymerase that catalyzes biosynthesis of RNA from an RNA template.  RNA replication helps the RNA viruses easily reproduce their progeny viruses.