RNA Metabolism: Synthesis, Polymerase and Catabolism

In this article we will discuss about:- 1. RNA Synthesis 2. RNA Polymerase 3. Sigma (σ) Factors 4. Ribozymes 5. Catabolism 6. Ribosomal RNAs are Processed from a Large Precursor 7. Messenger RNA (mRNA) is Modified at the 5′ and 3′ Ends 8. Transfer RNA (tRNA) is Extensively Processed and Modified.

Contents:

RNA Synthesis
RNA Polymerase
Sigma (σ) Factors of RNA
RNA also Knows as Ribozymes
Catabolism of RNA
Ribosomal RNAs are Processed from a Large Precursor
Messenger RNA (mRNA) is Modified at the 5′ and 3′ Ends
Transfer RNA (tRNA) is Extensively Processed and Modified

1. RNA Synthesis:

i. Transcription is the process by which the synthesis of RNA molecule is initiated and terminated.

ii. Stages of Transcription:

(a) Formation of transcription complex:

The enzyme RNA polymerase (DNA dependent) needs to bind with specific sequences on a DNA. The sequences recognised by RNA polymerase are called promoter. The DNA strand that serves as template for RNA synthesis is called template strand (-strand).

The DNA strand complementary to the template is called the non-template strand (+ strand). This non-template strand is called coding strand. RNA polymerase (holoenzyme) consists of core enzyme and sigma (a) factor.

Sigma factor recognises the promoter sequences.

RNA polymerase attaches to promoter region.

RNA polymerase melts the helical structure and separates 2 strands of DNA locally.

RNA polymerase initiates RNA synthesis. The site at which the first nucleotide is incorporated is called the start point.

(b) Initiation:

Core enzyme starts transcription at the separated DNA strand of an initiation complex. The subunit of RNA polymerase has two specific binding sites for the binding of nucleotide triphosphate (NTP). The first incoming NTP binds to RNA polymerase at the start point of initiation site and H bonds to the complementary base on the DNA within complex.

This site binds only purine nucleotide triphosphate either A or G. The binding is with 3′ end of the NTP leaving 5′ end to be free. The second incoming NTP binds to the elongation site on the polymerase.

This dinucleotide tetra-phosphate has either as the 5′ terminal nucleotide. After this phosphodiester bond formation a factor is released. First base is then dissociated from the initiation site and indicates the completion of initiation.

(c) Elongation:

The core enzyme polymerase moves in 3′-5′ direction of the coding strand and it adds successive NTPs at the 3′-OH end of the ribonucleotide chain already laid down in 5′-3′ direction. The incoming NTP forms a phosphodiester bond with 3′-OH end of the proceeding ribonucleotide. The DNA helix recloses after RNA polymerase transcribes through it and growing RNA chain dissociates from the DNA.

(d) Termination:

Termination of the synthesis of the RNA molecule is signaled by a sequence in the template strand of the DNA molecule, a signal that is recognized by a termination protein, Rho (p) factor. At the specific termination sites the new RNA chain is released.

After the termination, core enzyme separates from the DNA template and the core enzyme again binds with a factor and takes part in the formation of new RNA molecule. After the end of termination, Rho (p) factor dissociates RNA and this factor is again recycled. The overall process is given in Fig. 25.12.

2. RNA Polymerase:

i. It is the key enzyme for transcription.

ii. A single type of RNA polymerase is responsible for the synthesis of mRNA, rRNA, tRNA etc.

iii. Different enzymes are required to synthesize different types of RNA. They are called RNA polymerase I, RNA polymerase II, III, etc.

iv. Subunits composition of RNA polymerase of E. Coli.

v. The holoenzyme α₂ββ’σ can be separated into two components, the core enzyme (α₂ββ’) and the sigma factor.

vi. The holoenzyme requires template of double stranded DNA, four ribonucleotide triphosphates (GTP, ATP, UTP, CTP) and Mg⁺⁺ or Mn⁺⁺.

vii. Holoenzyme initiates transcription.

viii. Core enzyme has the ability to synthesize RNA on a DNA template.

3. Sigma (σ) Factors of RNA:

Transcription initiation requires specificity proteins (known as σ factors). These sigma factors bind reversibly to the catalytically active core RNA polymerase.

The core RNA polymerase interacts with sigma (σ) factors to form a holoenzyme. After the release of σ factor the core enzyme elongates an RNA chain.

The majority of cellular transcription requires the primary σ factor. In E. coli the primary σ factor is referred to as σ⁷⁰ and in B. subtilis σ⁴³.

In recent years, the genes encoding bacterial and phage a factors have been isolated and their amino acid sequences have been deduced from the corresponding DNA sequences. This shows that many σ factors have similar amino acid sequences and form a homologous protein family, σ factors thus may use a variety of protein structures to perform related functions in bacterial cells.

Biochemical Activities of σ Factors:

The biochemical activities of bacterial σ factors include:

(a) Core binding

(b) Activation of promoter recognition

(d) Inhibition of nonspecific transcription.

(e) All σ factors have at least the first two of these activities.

Different σ factors interact with the same region of the core RNA polymerase complex. Therefore, many o factors may share a common protein structure that allows core binding. Promoter recognition can occur only when holoenzyme is formed.

It seems most likely that σ factors themselves make sequence-specific contacts with promotion. Bacterial σ factors do not bind lightly to DNA in the absence of core RNA polymerase. As with promoter recognition, σ factors do not show a DNA melting activity in the absence of the core RNA polymerase subunits.

Lastly, the E. coli σ⁷⁰ factors have been shown to reduce the affinity of the core subunits for non- promoter containing DNA promoter by about 10⁴. It has been argued that this activity is important in allowing promoter recognition against large background of non-specific DNA. In B. subtilis, a separate protein component, δ²¹, inhibits core-mediated transcription of non-promoter containing DNA.

The δ²¹ protein binds to the B. subtilis core enzyme in the presence and absence of σ factors and reduces the interaction of RNA polymerase with the DNA template. Interestingly, the ability to inhibit nonspecific transcription has also been attributed to a specificity factor from yeast mitochondria that may be functionally similar to a σ factor.

General Features of σ Factors:

(a) σ factors form a heterogeneous group of proteins.

(b) E.coli σ⁷⁰ and B. subtilis σ⁴³ have approximately 50% amino acid identity allowing for a 245-amino acid gap introduced into the smaller σ⁴³ factor.

(c) All the related σ proteins are acidic with an excess of negative charge at pH 7.0. E. coli σ⁷⁰ factor is one of the most acidic proteins in E. coli.

(d) Most bacterial a factors are single polypeptides.

4. RNA also Knows as Ribozymes:

a. The RNAs which show highly substrate- specific catalytic activity and meet all the classic criteria for definition as enzymes are termed ribozymes.

b. They catalyse trans-esterification and ultimately hydrolysis of phosphodiester bonds in RNA molecules. These reactions are facilitated by free -OH groups e.g., on guanosyl residues.

c. They play key roles in the intron splicing events essential for the conversion of Pre-mRNAs to mature mRNAs.

d. Recently, a ribosomal RNA component had been noted to hydrolyze an aminoacyl ester and thus to play a central role in peptide bond function. These observations, made in organelles from plants, yeast, viruses, and higher eukaryotic cells, show that RNA can act as an enzyme.

5. Catabolism of RNA:

a. Ribonucleases specifically hydrolyze ribonucleic acids.

b. Endonucleases cleave internal phosphodiester bonds to produce a 3′-hydroxyl and a 5′-phospharyl or a 5′-hydroxyl and a 3′-phospharyl terminus.

c. Exonucleases are capable of hydrolysing a nucleotide only when it is present at a terminus of a molecule.

RNA Molecules are often Processed before they become Functional:

i. All eukaryotic RNA primary transcripts undergo processing before their function whether it be as mRNA or rRNA or tRNA. This processing occurs primarily within the nucleus. The processing includes nucleolytic cleavage to smaller molecules and coupled nucleolytic and ligation reactions.

ii. In mammalian cells, 50-75 per cent of the nuclear RNA does not contribute to the cytoplasmic mRNA. This nuclear RNA loss is significantly greater than the loss of intervening sequences alone.

The Coding Portions (Exons) of most Eukaryotic Genes are Interrupted by Introns:

i. The exons of many genes are long sequences of DNA that do not supply the genetic information and are finally translated into the amino acid sequence of a protein molecule. These sequences interrupt the coding region of structural genes. These intervening sequences (introns) exist in most eukaryotes.

ii. The intron RNA sequences are cleaved out of the transcript and the exons of the transcript are spliced together in the nucleus before the resulting mRNA molecule appears in the cytoplasm for translation. Exons are the means of supplying genetic information.

Introns are Removed and Exons are Spliced together:

i. After removal of the introns, the exons are ligated to form the mRNA molecule which is transported to the cytoplasm.

ii. Of the four different splicing reaction mechanisms, the one most frequently used in eukaryotic cells is described here. There are reasonably conserved sequences at each of the two exon-intron (splice) junctions and at the branch site which is located 20-40 nucleotides upstream from the 3′ splice site. The spliceosome, a special structure, is involved in converting the primary transcript into mRNA.

iii. Spliceosomes consist of the primary transcript, five small nuclear RNAs (U1, U2, U5, and U4/U6) and more than 50 proteins. Collectively, these form a small nucleoprotein (snRNP) complex, sometimes called a snurp. Snurps are used to position the RNA segments for the necessary splicing reactions.

The splicing reaction starts with a cut at the junction of the 5′ exon. This is performed by a nucleophilic attack by an adenylyl residue in the branch point sequence located just upstream from the 3′ end of this intron.

iv. The free 5′ terminal then forms a loop which is linked by an unusual 5′ — 2′ phosphodiester bond to the reactive A in the PyNPyPyPuAPy branch site sequence (Fig. 25.13). The adenylyl residue is typically loated 28-37 nucleotides upstream from the 3′ end of the intron being removed. The branch site identifies the 3′ splice site.

A second cut is made at the junction of the intron with the 3′ exon. In this second trans-esterification reaction, the 3′ hydroxyl of the upstream exon attacks the 5′ phosphate at the downstream exon- intron boundary and the structure containing the intron is released and hydrolyzed. The 5′ and 3′ exons are ligated to form a continuous sequence.

v. U1 binds first to the 5 exon-intron boundary by base pairing U2 then binds, by base pairing, to the branch site. A U5/U4/U6 complex next joins the spliceosome. An ATP-dependent protein-mediated unwinding results in the disruption of the base paired U4/U6 complex with the release of U4. U6 is then able to interact first with U2, then with U1.

These interactions serve to approximate the 5′ splice site, the branch point with its reactive A, and then 3′ splice site. This is en-chanced by U5. The two ends are cleaved by the U2/U6 complex. RNA serves as a catalytic agent. This sequence is then repeated in genes containing multiple introns. The introns are not necessarily removed in sequence — 1, then 2, then 3, etc.

vi. The hn RNA molecules are the primary transcripts plus their early processed products which are transported to the cytoplasm as mature mRNA molecules after the addition of Caps and Poly (A) tails and removal of the portion corresponding to the introns.

6. Ribosomal RNAs are Processed from a Large Precursor:

i. In mammalian cells, three rRN A molecules are transcribed to act as large precursor molecule. The precursor is then processed in the nucleolus to provide the RNA component for the ribosome subunits of the cytoplasm.

The rRNA genes are present in the nucleoli and hundreds of these genes are present in every cell. These large number of genes ultimately synthesize many copies of each type of rRNA to form the 10⁷ ribosomes required for each cell replication. A single mRNA molecule is copied into 10⁵ protein molecules, providing a large amplification, the rRNAs are end products.

ii. Each of units being transcribed by the rRNA genes encodes (5′ to 3′) an 18s, a 5.8s and a 28s ribosomal RNA. The primary transcript is a 45 s molecule which is highly methylated in the nucleolus. In the 45s precursor, the 28s segment contains 65 ribose-methyl groups and 5 base-methyl groups. The 45s precursor is nucleolytically processed.

iii. During the processing of rRNA further methylation occurs. In the nucleoli, the larger 60s subunit is formed by the assembling of 28s chains with 50 ribosomal proteins. The 5.8s rRNA molecule is also formed from the 45 s precursor RNA in the nucleolus. The 18s rRNA molecule is associated with 30 ribosome proteins to form the ribosomal subunits (40s).

7. Messenger RNA (mRNA) is Modified at the 5′ and 3′ Ends:

i. Mammalian mRNA molecules contain a 7-methylguanosine cap structure at their 5 terminal, and most have a poly (A) tail at the 3′ terminal. The cap structure is added to the 5′ end of the newly transcribed mRNA precursor in the nucleus prior to transport of the mRNA molecule to the cytoplasm.

The 5′ cap of the RNA transcript is necessary for translation initiation and protection of the 5′ end of mRNA from attack by 5′ → 3′ exonucleases.

ii. Poly (A) tails are added to the 3′ end of mRNA molecules in the post transcriptional processing step. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence. Poly (A) polymerase adds a short poly (A) tail which is extended to 200 A residues.

This enzyme protects the 3′ end of mRNA from 3′ → 5′ exonuclease attack. In mammalian cells, cytoplasmic processes can add and remove adenylate residues from the poly (A) tails.

iii. The extra nucleotides occur in noncoding regions both 5′ and 3′ to the coding region, the longest noncoding sequences are usually at the 3′ end.

8. Transfer RNA (tRNA) is Extensively Processed and Modified:

i. The tRNA molecules act as adaptor molecules for the translation of mRNA into protein sequences. The tRN As are reduced in size by a specific class of ribonucleases. The genes of some tRNA molecules contain a single intron 10—40 nucleotides long.

These introns are transcribed. The processing of the precursor transcripts of many tRNA molecules must include removal of the introns and proper splicing of the anticodon region to generate an active adaptor molecule for protein synthesis.

ii. The further modification of the tRNA molecules includes nucleotide alkylation’s and the attachment of the characteristic C-C-A terminal at the 3′ end of the molecule by the enzyme nucleotidyl transferase. The 3’OH of the A ribose is the point of attachment for the specific amino acid that is to enter the polymerization reaction of protein synthesis.

The methylation of mammalian tRNA precursors occurs in the nucleus, but the cleavage and attachment of C-C-A are cytoplasmic functions. The attachment of amino acids to the C-C-A residues require the enzymes within the cytoplasm of mammalian cells.