RNA SYNTHESIS

Transcription is the mechanism by which a template strand of DNA is utilized by specific RNA polymerases to generate one of the three different classifications of RNA. These 3 RNA classes are:

 

• 1. Messenger RNAs (mRNAs): This class of RNAs are the genetic coding templates used by the translational machinery to determine the order of amino acids incorporated into an elongating polypeptide in the process of translation.
• 2. Transfer RNAs (tRNAs): This class of small RNAs form covalent attachments to individual amino acids and recognize the encoded sequences of the mRNAs to allow correct insertion of amino acids into the elongating polypeptide chain.
• 3. Ribosomal RNAs (rRNAs): This class of RNAs are assembled, together with numerous ribosomal proteins, to form the ribosomes. Ribosomes engage the mRNAs and form a catalytic domain into which the tRNAs enter with their attached amino acids. The proteins of the ribosomes catalyze all of the functions of polypeptide synthesis.

 

All RNA polymerases are dependent upon a DNA template in order to synthesize RNA. The resultant RNA is, therefore, complimentary to the template strand of the DNA duplex and identical to the non-template strand. The non-template strand is called the coding strand because its' sequences are identical to those of the mRNA. However, in RNA, U is substituted for T.

 

 

Classes of RNA Polymerases

 

In prokaryotic cells, all 3 RNA classes are synthesized by a single polymerase. In eukaryotic cells there are 3 distinct classes of RNA polymerase, RNA polymerase (pol) I, II and III. Each polymerase is responsible for the synthesis of a different class of RNA. The capacity of the various polymerases to synthesize different RNAs was shown with the toxin a-amanitin. At low concentrations of a-amanitin synthesis of mRNAs are affected but not rRNAs nor tRNAs. At high concentrations, both mRNAs and tRNAs are affected. These observations have allowed the identification of which polymerase synthesizes which class of RNAs. RNA pol I is responsible for rRNA synthesis (excluding the 5S rRNA).There are 4 major rRNAs in eukaryotic cells designated by there sedimentation size. The 28S, 5S 5.8S RNAs are associated with the large ribosomal subunit and the 18S rRNA is associated with the small ribosomal subunit. RNA pol II synthesizes the mRNAs and some of the small nuclear RNAs (snRNAs) involved in RNA splicing. RNA pol III synthesizes the tRNAs, the 5S rRNA and some snRNAs.

Mechanism of RNA Polymerases

 

Synthesis of RNA exhibits several features that are synonymous with DNA replication. RNA synthesis requires accurate and efficient initiation, elongation proceeds in the 5' ---> 3' direction (i.e. the polymerase moves along the template strand of DNA in the 3' ----> 5' direction), and RNA synthesis requires distinct and accurate termination. Transcription exhibits several features that are distinct from replication.

 

1. Transcription initiates, both in prokaryotes and eukaryotes, from many more sites than replication.

• 2. There are many more molecules of RNA polymerase per cell than DNA polymerase.
• 3. RNA polymerase proceeds at a rate much slower than DNA polymerase (approximately 50-100 bases/sec for RNA versus near 1000 bases/sec for DNA).
• 4. Finally the fidelity of RNA polymerization is much lower than DNA. This is allowable since the aberrant RNA molecules can simply be turned over and new correct molecules made.

Processes of Transcription

 

Signals are present within the DNA template that act in cis to stimulate the initiation of transcription. These sequence elements are termed promoters. Promoter sequences promote the ability of RNA polymerases to recognize the nucleotide at which initiation begins. Additional sequence elements are present within genes that act in cis to enhance polymerase activity even further. These sequence elements are termed enhancers. Transcriptional promoter and enhancer elements are important sequences used in the control of gene expression.

E. coli RNA polymerase is composed of 5 distinct polypeptide chains. Association of several of these generates the RNA polymerase holoenzyme. The sigma subunit is only transiently associated with the holoenzyme. This subunit is required for accurate initiation of transcription by providing polymerase with the proper cues that a start site has been encountered.

In both prokaryotic and eukaryotic transcription the first incorporated ribonucleotide is a purine and it is incorporated as a triphosphate. In E. coli several additional nucleotides are added before the sigma subunit dissociates.

Elongation involves the addition of the 5'-phosphate of ribonucleotides to the 3'-OH of the elongating RNA with the concomitant release of pyrophosphate. Nucleotide addition continues until specific termination signals are encountered. Following termination the core polymerase dissociates from the template. The core and sigma subunit can then reassociate forming the holoenzyme again ready to initiate another round of transcription.

In E. coli transcriptional termination occurs by both factor-dependent and factor-independent means. Two structural features of all E. coli factor-independently terminating genes have been identified. One feature is the presence of 2 symmetrical GC-rich segments that are capable of forming a stem-loop structure in the RNA and the second is a downstream A rich sequence in the template. The formation of the stem-loop in the RNA destabilizes the association between polymerase and the DNA template. This is further destabilized by the weaker nature of the AU base pairs that are formed, between the template and the RNA, following the stem-loop. This leads to dissociation of polymerase and termination of transcription. Most genes in E. coli terminate by this method.

Factor-dependent termination requires the recognition of termination sequences by the termination protein, rho. The rho factor recognizes and binds to sequences in the 3' portion of the RNA. This binding destabilizes the polymerase-template interaction leading to dissociation of the polymerase and termination of transcription.

 

Posttranscriptional Processing of RNAs

 

When transcription of bacterial rRNAs and tRNAs is completed they are immediately ready for use in translation. No additional processing takes place. Translation of bacterial mRNAs can begin even before transcription is completed due to the lack of the nuclear-cytoplasmic separation that exists in eukaryotes. The ability to initiate translation of prokaryotic RNAs while transcription is still in progress affords a unique opportunity for regulating the transcription of certain genes. An additional feature of bacterial mRNAs is that most are polycistronic. This means that multiple polypeptides can be synthesized from a single primary transcript. This does not occur in eukaryotic mRNAs.

In contrast to bacterial transcripts, eukaryotic RNAs (all 3 classes) undergo significant post-transcriptional processing. All 3 classes of RNA are transcribed from genes that contain introns. The sequences encoded by the intronic DNA must be removed from the primary transcript prior to the RNAs being biologically active. The process of intron removal is called RNA splicing. Additional processing occurs to mRNAs. The 5' end of all eukaryotic mRNAs are capped with a unique 5' -----> 5' linkage to a 7-methylguanosine residue. The capped end of the mRNA is thus, protected from exonucleases and more importantly is recognized by specific proteins of the translational machinery.

Structure of the 5'-Cap of Eukaryotic mRNAs

 

 

 

Messenger RNAs also are polyadenylated at the 3' end. A specific sequence, AAUAAA, is recognized by the endonuclease activity of by polyadenylate polymerase which cleaves the primary transcript approximately 11 - 30 bases 3' of the sequence element. A stretch of 20 - 250 A residues is then added to the 3' end by the polyadenylate polymerase activity.

 

Processes of Polyadenylation

 

 

 

In addition to intron removal in tRNAs, extra nucleotides at both the 5' and 3' ends are cleaved, the sequence 5'-CCA-3' is added to the 3' end of all tRNAs and several nucleotides undergo modification. There have been more than 60 different modified bases identified in tRNAs.

Both prokaryotic and eukaryotic rRNAs are synthesized as long precursors termed preribosomal RNAs. In eukaryotes a 45S preribosomal RNA serves as the precursor for the 18S, 28S and 5.8S rRNAs.

Splicing of RNAs

 

There are 4 different classes of introns. The 2 most common are the group I and group II introns. Group I introns are found in nuclear, mitochondrial and chloroplast rRNA genes, group II in mitochondrial and chloroplast mRNA genes. Many of the group I and group II introns are self-splicing, i.e. no additional protein factors are necessary for the intron to be accurately and efficiently spliced out.

Group I introns require an external guanosine nucleotide as a cofactor. The 3'-OH of the guanosine nucleotide acts as a nucleophile to attack the 5'-phosphate of the 5' nucleotide of the intron. The resultant 3'-OH at the 3' end of the 5' exon then attacks the 5' nucleotide of the 3' exon releasing the intron and covalently attaching the two exons together. The 3' end of the 5' exon is termed the splice donor site and the 5' end of the 3' exon is termed the splice acceptor site.

 

 

Group 1 Self-Splicing Introns

 

 

 

Process of Splicing by Group 1 Introns

Group II introns are spliced similarly except that instead of an external nucleophile the 2'-OH of an adenine residue within the intron is the nucleophile. This residue attacks the 3' nucleotide of the 5' exon forming an internal loop called a lariat structure. The 3' end of the 5' exon then attacks the 5' end of the 3' exon as in group I splicing releasing the intron and covalently attaching the two exons together.

Group 2 Self-Splicing Introns

 

 

 

 

Processes of Splicing by Group 2 Introns

 

 

 

The third class of introns is also the largest class found in nuclear mRNAs. This class of introns undergoes a splicing reaction similar to group II introns in that an internal lariat structure is formed. However, the splicing is catalyzed by specialized RNA-protein complexes called small nuclear ribonucleoprotein particles (snRNPs, pronounced snurps). The RNAs found in snRNPs are identified as U1, U2, U4, U5 and U6. The genes encoding these snRNAs are highly conserved in vertebrate and insects and are also found in yeasts and slime molds indicating their importance.

Analysis of a large number of mRNA genes has led to the identification of highly conserved consensus sequences at the 5' and 3' ends of essentially all mRNA introns.

 

 

 

The U1 RNA has sequences that are complimentary to sequences near the 5' end of the intron. The binding of U1 RNA distinguishes the GU at the 5' end of the intron from other randomly placed GU sequences in mRNAs. The U2 RNA also recognizes sequences in the intron, in this case near the 3' end. The addition of U4, U5 and U6 RNAs forms a complex identified as the spliceosome that then removes the intron and joins the two exons together.

The fourth class of introns are those found in certain tRNAs. These introns are spliced by a specific splicing endonuclease that utilizes the energy of ATP hydrolysis to catalyze intron removal and ligation of the two exons together.

 

 

Clinical Significances of Alternative and Aberrant Splicing

 

The presence of introns in eukaryotic genes would appear to be an extreme waste of cellular energy when considering the number of nucleotides incorporated into the primary transcript only to be removed later as well as the energy utilized in the synthesis of the splicing machinery. However, the presence of introns can protect the genetic makeup of an organism from genetic damage by outside influences such as chemical or radiation. An additionally important function of introns is to allow alternative splicing to occur, thereby, increasing the genetic diversity of the genome without increasing the overall number of genes. By altering the pattern of exons, from a single primary transcript, that are spliced together different proteins can arise from the processed mRNA from a single gene. Alternative splicing can occur either at specific developmental stages or in different cell types.

This process of alternative splicing has been identified to occur in the primary transcripts from at least 40 different genes. Depending upon the site of transcription, the calcitonin gene yields an RNA that synthesizes calcitonin (thyroid) or calcitonin-gene related peptide (CGRP, brain). Even more complex is the alternative splicing that occurs in the a-tropomyosin transcript. At least 8 different alternatively spliced a-tropomyosin mRNAs have been identified.

Abnormalities in the splicing process can lead to various disease states. Many defects in the b-globin genes are known to exist leading to b-thalassemias. Some of these defects are caused by mutations in the sequences of the gene required for intron recognition and, therefore, result in abnormal processing of the b-globin primary transcript.

Patients suffering from a number of different connective tissue diseases exhibit humoral auto-antibodies that recognize cellular RNA-protein complexes. Patients suffering from systemic lupus erythematosis have auto-antibodies that recognize the U1 RNA of the spliceosome.