1. RNA Structure, synthesis, and processing

2. Structure of RNA
There are three major types of RNA that participate in the process of protein synthesis: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). Like DNA, these types of RNA are unbranched polymeric molecules composed of nucleoside monophosphates joined together by phosphodiester bonds and their pentose sugar moiety is ribose.

3. In general, types of RNA differ as a group from DNA in many aspects:
They are considerably smaller than DNA.
They contain ribose instead of deoxyribose and uracil instead of thymine.
Unlike DNA, most RNAs exist as single strands that are capable of folding into complex structures.
[Note: In eukaryotes, small RNA molecules found in the nucleus (snRNAs) perform specialized functions].

4. [Note: Some rRNA function as catalysts in protein synthesis]
A. Ribosomal RNA (r RNA)
rRNAs are present in association with several proteins as structural components of the ribosomes—the sites for protein synthesis. In prokaryotic cells, there are three major species of rRNA (23S, 16S, and 5S), while in eukaryotes, there are four rRNA species (28S, 18S, 5.8S, and 5S). [Note: “S” is the Svedberg unit, which is related to the molecular weight and shape of the compound.] Together, rRNA make up about 80% of the total RNA in the cell.
[Note: Some rRNA function as catalysts in protein synthesis]

6. B. Transfer RNA
tRNA are the smallest (4S) of the three major species of RNA. There is at least one specific type of tRNA molecule for each of the twenty amino acids commonly found in proteins. Together, tRNA make up about 15% of the total RNA in the cell. There are 2 important features of tRNA:
They contain unusual bases e.g, dihydrouracil (D) and pseudouracil (ψ) .
They have extensive complementary intrachain base-pairing leading to extensive looping and folding giving rise to the secondary and tertiary structures of t-RNA..

7. Each tRNA can recognize the signal or the message on the mRNA by its anticodon sequence (anticodon loop), so that it brings up the specific amino acid covalently attached to its 3′-end to add it to the growing polypeptide chain.

9. C. Messenger RNA
It comprises only about 5% of the RNA in the cell. mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis. It is widely different in size and base sequences. It contains coding regions to be translated in addition to other untranslated regulatory regions at both 5′- and 3′-ends. Special structural characteristics of eukaryotic mRNA include a long sequence of adenine nucleotides (a “poly-A tail”) on the 3′-end of the RNA chain, plus a “cap” on the 5′-end consisting of a molecule of 7-methylguanosine attached “backward” (5′→5′) through a triphosphate linkage.

11. Transcription of Eukaryotic Genes
The transcription of eukaryotic genes is a far more complicated process than transcription in prokaryotes. There are specific separate polymerases for the synthesis of rRNA, tRNA, and mRNA. In addition, a number of proteins called transcription factors are involved. These factors have two main functions:
1. They recognize which genes are to be transcribed.
They help in the assembly of the transcription complex at the promoter region of that gene.
[Note: Each eukaryotic RNA polymerase has its own promoters and transcription factors].
For transcription factors to recognize and bind to their specific DNA sequences, the chromatin structure in that region must be altered to allow access to the DNA.

12. A major mechanism by which chromatin is remodeled is through acetylation of lysine residues at the amino terminus of histone proteins. This process of opening up the chromatin and making DNA more accessible for transcription is mediated by histone acetyltransferases. Removal of the acetyl group by histone deacetylases restores the positive charge, and fosters stronger interactions between histones and DNA.

13. There are 3 classes of RNA polymerase in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each class of RNA polymerase recognizes particular types of genes:
RNA polymerase I: It synthesizes the precursor of the 28S, 18S, and 5.8S r-RNA in the nucleolus.
RNA polymerase II: It synthesizes the precursors of m-RNA in addition to snRNA.
RNA polymerase III: It produces the small RNA, including tRNA, 5S ribosomal RNA, and some snRNA.

14. Steps of RNA Synthesis (DNA Transcription)
The process of transcribing a typical gene in an eukaryotic cell is divided into 3 phases; initiation, elongation and termination.
Initiation: begins with binding of RNA polymerase to a region in the DNA known as the promoter. In eukaryotic cells, each class of polymerases has specific transcription factors and specific promotor areas. When a DNA sequence in a specific gene is to be transcribed to mRNA using RNA polymerase II, the ‘promoter’ consensus sequence is called TATA or Hogness box that is centered about 25 nucleotides to the left of the transcription start site. Another consensus sequence is found between 70 and 80 nucleotides to the left as well and called CAAT box.

16. In some eukaryotic genes, TATA box is substituted by a GC-rich region (GC box). Such sequences in the promoter area serve as binding sites for proteins known as transcription factors, which in turn interact with each other and with RNA polymerase II. Eukaryotic RNA polymerase II does not itself recognize and bind the promoter, but is brought to the promoter by the general transcription factor, TFIIF. In addition to binding DNA, specific transcription factors also bind other proteins (“coactivators”), recruiting them to the transcription complex.
[Note: Coactivators include the histone acetyltransferase proteins involved in chromatin remodeling]

17. In order to enhance this step (initiation), there are probably thousands of specific DNA sequences away from the transcription start site (toward both 5′- and 3′-ends) called as Enhancers or Response elements that bind to specific proteins called Activators which interact with the Transcription complex to stimulate the transcription.

18. 2. Elongation:
Once the promoter region has been recognized and bound by the transcription complex, local unwinding (melting) of DNA helix occurs. The process generates supercoils in the DNA that can be relieved by (DNA topoisomerases I and II). RNA polymerase II begins to synthesize a transcript of the DNA sequence. The elongation phase is said to begin when the transcript (typically starting with a purine) exceeds 10 nucleotides in length and the enzyme starts to leave the promoter region and moves along the template DNA strand in 5′ → 3′ processive manner. During replication, a short DNA-RNA hybrid helix is formed.

20. 3. Termination:
The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (spontaneous) or dependent upon the participation of a protein known as the ρ (rho) factor. This leads to stop transcription and the DNA double helix to zip up again.

21. Posttranscriptional Modification of RNA
The RNA strand that has been produced from DNA transcription is known as the “primary transcript”. It is a linear RNA copy of the transcriptional unit (i.e DNA segment between the specific initiation and the termination sequences). The primary transcript of rRNA and tRNA are transcriptionally modified by cleavage of the original transcript by ribonucleases. tRNA will be further modified to give each species its unique identity. mRNA primary transcript will be extensively modified as well.

22. r-RNA of both prokaryotic and eukaryotic cells are synthesized from long precursor molecules called (preribosomal RNAs). The 28S, 18S, and 5.8S rRNA of eukaryotes are produced by the cleavage of the primary transcript by ribonuclease enzyme.

24. Transfer RNA posttranscriptional modification
Functional t-RNA is produced from a longer precursor molecule (pre- t RNA) after modification. An intron (14 nucleotide intron) must be removed from the anticodon loop, and sequences at both the 5′- and the 3′-ends of the molecule must be trimmed. Other posttranscriptional modifications include addition of CCA sequence by nucleotidyltransferase to the 3′-terminal end of tRNA, and modification of bases at specific positions to produce “unusual bases” like (D= Dihydrouracil, ψ= pseudouracil, and m= methylated bases).
Note: the CCA sequence on the 3′-end of t-RNA is present in all mature or functional t-RNA species.

26. Eukaryotic mRNA posttranscriptional modification
The mRNA molecule synthesized in eukaryotic nuclei by RNA polymeraseII is a collection of the precursor molecules of mRNA called as heterogeneous nuclear RNA (hnRNA). The primary transcripts are extensively modified in the nucleus after transcription. These modifications usually include:
1. 5′ “Capping”: This process is the first of the processing reactions for hnRNA The cap is a 7-methylguanosine attached “backward” to the 5′-terminal end of the mRNA, forming an unusual 5′→5′ triphosphate linkage. The creation of the guanosine triphosphate part of the cap requires the nuclear enzyme guanylyltransferase. Methylation of this terminal guanine occurs in the cytosol, and is catalyzed by guanine-7-methyltransferase.

28. the presence of this 7- methyl guanosine triphosphate cap is very essential in starting the mRNA translation later on (i.e. protein synthesis).

29. 2. Addition of a poly-A tail
Most eukaryotic mRNA have a chain of 40–200 adenine nucleotides attached to the 3′-end of mRNA primary transcript. This poly-A tail is not transcribed from the DNA, but rather is added after transcription by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate. The mRNA is cleaved downstream of a consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3′-end of the RNA, and the poly-A tail is added to the new 3′-end. These tails help stabilize the mRNA and facilitate their exit from the nucleus. After the mRNA enters the cytosol, the poly-A tail is gradually shortened.

30. 3. Removal of introns
Maturation of eukaryotic mRNA usually involves the removal of RNA sequences, which do not code for protein (introns, or intervening sequences) from the primary transcript. The remaining coding sequences, the exons, are joined together to form the mature mRNA. The process of removing introns and joining exons is called splicing. The molecular machine that accomplishes these tasks is known as the spliceosome.