MBC 224

TRANCRIPTION
INTRODUCTION

 The synthesis of an RNA molecule from DNA is a complex process involving one of the group of
RNA polymerase enzymes and a number of associated proteins.

 The general steps required to synthesize the primary transcript are initiation, elongation, and
termination.

 The RNA molecules synthesized in mammalian cells are made as precursor molecules that have to
be processed into mature, active RNA.

 All eukaryotic cells have four major classes of RNA:

 ribosomal RNA (rRNA)
 messenger RNA (mRNA), transfer RNA (tRNA)
 small nuclear RNA (snRNA).

The first three are involved in protein synthesis, and snRNA is involved in mRNA splicing
 SYNTHESIS OF RNA

• The processes of DNA and RNA synthesis are similar in the following ways:

 the general steps of initiation, elongation, and termination with 5′ to 3′ polarity

 large, multicomponent initiation complexes

 adherence to Watson-Crick base-pairing rules.

• The synthesis of DNA and RNA differ in the following ways:

 Ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides.

 Uracil replaces thymine as the complementary base pair for A in RNA.

 A primer is not involved in RNA synthesis.

 Only a very small portion of the genome is transcribed or copied into RNA, whereas the entire
genome must be copied during DNA replication.

 There is no proofreading function during RNA transcription.

 The strand that is transcribed or copied into an RNA molecule is referred to as the template strand
of the DNA.

 The other DNA strand is known as the coding strand of that gene because, with the exception of T
for U changes, it corresponds exactly to the sequence of the primary transcript, which encodes the
protein product of the gene.

 The information in the template strand is read out in the 3′ to 5′ direction.

 DNA-dependent RNA polymerase is the enzyme responsible for the polymerization of

ribonucleotides into a sequence complementary to the template strand of the gene.

 The enzyme attaches at a specific site called the promoter on the template strand. This is followed
by initiation of RNA synthesis at the starting point, and the process continues until a termination
sequence is reached.

 A transcription unit is defined as that region of DNA that includes the signals for transcription
initiation, elongation, and termination.
 The RNA product that is synthesized in the 5′ to 3′ direction, is the primary transcript. In
prokaryotes, this can represent the product of several contiguous genes but in mammalian cells, it
usually represents the product of a single gene.

 The 5′ terminals of the primary RNA transcript and the mature cytoplasmic RNA are identical.
Hence, the starting point of transcription corresponds to the 5 nucleotide of the mRNA. This is
designated position +1, as is the corresponding nucleotide in the DNA.

 The primary transcripts generated by RNA polymerase II in eukaryotes are readily capped by 7-
methylguanosine triphosphate caps that remains and eventually appear on the 5′ end of mature
cytoplasmic mRNA.

 These caps help in the subsequent processing of the primary transcript to mRNA, for the
translation of the mRNA, and for protection of the mRNA against exonucleolytic attack.
 TYPES OF RNA POLYMERASES

S/N TYPE OF RNA POLYMERASE RNA PRODUCT

1 RNA Polymerase I rRNA

2 RNA Polymerase II mRNA

3 RNA Polymerase III tRNA/SSrRNA

 Each of these DNA-dependent RNA polymerases is responsible for transcription of different sets of genes.

 The sizes of the RNA polymerases range from MW 500,000 to MW 600,000. These enzymes are much more
complex than prokaryotic RNA polymerases.

 They all have two large subunits and a number of smaller subunits as many as 14 in the case of RNA pol III.

 The eukaryotic RNA polymerases have extensive amino acid homologies with prokaryotic RNA
polymerases.

 The functions of each of the subunits are not yet fully understood. Many could have regulatory functions,
such as serving to assist the polymerase in the recognition of specific sequences like promoters and
termination signals.

 The peptide toxin, α-amanitin from the mushroom Amanita phalloides is an inhibitor used to differentiate
the different eukaryotic nuclear DNA-dependent RNA polymerases.

 α-Amanitin blocks the translocation of RNA polymerase during transcription

 RNA polymerase I is insensitive to α-Amanitin.

 RNA polymerase II is very sensitive to α-Amanitin.

 RNA polymerase III is moderately sensitive to α-Amanitin.

 Trancription starts with the binding of the RNA holopolymerase molecule to the template at the promoter site to
form a PIC.

 Binding is followed by a conformational change of the RNAP, and the first nucleotide (almost always a purine)
then associates with the initiation site on the β subunit of the enzyme.

 In the presence of the appropriate nucleotide, the RNAP catalyzes the formation of a phosphodiester bond, and
the nascent chain is now attached to the polymerization site on the β subunit of RNAP.

 Initiation of formation of the RNA molecule at its 5′ end then follows, while elongation of the RNA molecule from
the 5′ to its 3′ end continues cyclically, antiparallel to its template.
 The enzyme polymerizes the ribonucleotides in a specific sequence dictated by the template strand and
interpreted by Watson-Crick basepairing rules. Pyrophosphate is released in the polymerization reaction. This
pyrophosphate (PPi) is rapidly degraded to 2 mol of inorganic phosphate (Pi) by ubiquitous pyrophosphatases,
thereby providing irreversibility on the overall synthetic reaction. In both prokaryotes and eukaryotes, a purine
ribonucleotide is usually the first to be polymerized into the RNA molecule.
 In both prokaryotes and eukaryotes, 5′ triphosphate of the first nucleotide is maintained in mRNA. As the
elongation complex containing the core RNA polymerase progresses along the DNA molecule, DNA unwinding
must occur in order to provide access for the appropriate base pairing to the nucleotides of the coding strand.
The extent of this transcription bubble (ie, DNA unwinding) is constant throughout transcription and has been
estimated to be about 20 base pairs per polymerase molecule.

 The size of the unwound DNA region is determined by the polymerase and is independent of the DNA sequence
in the complex suggesting that RNA polymerase has associated with it an “unwindase” activity that opens the
DNA helix.
 Termination of the synthesis of the RNA molecule in bacteria is signaled by a sequence in the
template strand of the DNA molecule: a signal that is recognized by a termination protein, the rho
(ρ) factor.

 Rho is an ATP-dependent RNA-stimulated helicase that disrupts the nascent RNA-DNA complex.
After termination of synthesis of the RNA molecule, the enzyme separates from the DNA template
and probably dissociates to free core enzyme and free σ factor.

 With the assistance of another σ factor, the core enzyme then recognizes a promoter at which the
synthesis of a new RNA molecule commences.

 In eukaryotic cells, termination is less well defined. It seems to be associated both to initiation and
to addition of the 3′ polyA tail of mRNA and could involve destabilization of the RNA-DNA complex
at a region of A–U base pairs.
 POST TRANSCRIPTIONAL MODIFICATION

 The various processes that takes place after an RNA molecule has been transcribed
before the translation is called post-transcriptional modification.

 Post-transcriptional step can also be regulated to control gene expression in the cell.

 If the RNA is not processed, translocated, or translated, then no protein will be

synthesized.
 IMPORTANCE OF POST TRANSLATIONAL MODIFICATION

 Modifications facilitates the recognition of RNA molecule by molecules that mediate

RNA translation into proteins.

 During post-transcriptional processing, portions of the RNA chain that are not supposed
to be translated into proteins are excised from the sequence.

 Thus, post-transcriptional processing helps increase the efficiency of protein synthesis

by allowing only specific protein-coding RNA to go on to be translated.
 TYPES OF POST TRANSLATIONALMODIFICATION

• 5’ end capping
 Once the 5’ end of a nascent RNA extends free of the RNAP II
approximately 20-30 nt, it is ready to be capped by a 7-
methylguanosine structure.

 The 5’ cap functions as a recognition site for transport of the

completed mRNA out of the nucleus and into the cytoplasm.

 The process actually involves three steps. First, RNA triphosphatase

removes the 5’-terminal triphosphate group. Guanylation by GTP is
catalyzed by capping enzyme, forming an unusual 5’-5’ “backward”
bond between the new guanine and the first nucleotide of the RNA
transcript.

 Finally, guanine-7-methyltransferase methylates the newly

attached guanine.
3’-OH, Polyadenylation
 An enzyme complex that binds to a site on the CTD tail of RNAP II cleaves a portion of the 3’ end near an
AAUAAA recognition sequence and then serially adds a large number of adenine residues.

 The poly(A) tail is not required for translation, but it has an effect on the stability of transcripts in the
cytoplasm. As mRNA molecules stay in the cytoplasm 3 longer, the poly(A) tail is gradually removed.

 Once the poly(A) tail is gone, the mRNA will soon be destroyed. mRNA molecules with longer poly(A) tails
are survive longer in the cytoplasm than those with shorter tails, but there is currently no evidence for a
directly proportional effect.

 Although the enzyme that cleaves the primary transcript in preparation for polyadenylation has not been
identified, two nonenzymatic factors, the excitingly-named cleavage factor I (CFI) and cleavage factor II
(CFII) have been implicated.

 The serial adenylation comes from the activity of poly(A) polymerase (PAP) in conjunction with CPSF
(cleavage and polyadenylation speci city factor), which binds to the RNA.

 PAP itself has relatively poor affinity for RNA. As with other nucleic acid polymerases, it adds new
nucleotides onto the free 3’-OH of the pre-existing chain.
SPLICING

 Unlike prokaryotic RNA, which is a continuously translatable coding region immediately as it comes out
of the RNA polymerase, most eukaryotic RNAs have interrupted coding regions called INTRONS.

 Splicing is the process by which the non-coding regions (introns) are removed, and the coding regions,
known as exons, are connected together.
 In some RNAs, this can happen autonomously, with part of the RNA acting as an enzymatic catalyst for
the process. This requires that the RNA have a specific secondary and tertiary structure, bringing the two
exons close together while looping out the intron. It was the study of this phenomenon that led to the
discovery of ribozymes, which are enzymes made of RNA.

 Splicing removes some portions of a primary transcript (introns) while combining the remaining RNA
(exons) to form the final mRNA sequence.

 The chemical mechanism is a series of two transesterifications. The first accomplishes the looping of the
intron, while the second releases that intron while simultaneously joining the two exons together.

 The other two landmarks are the 3’ and the 5’ splice sites, each of which are sequences that bind to
snRNPs to bring together the spliceosome