RNA (ribonucleic acid)

Various Forms of RNA

Three major classes of cellular RNA molecules function during the expression of genetic information:
ribosomal RNA (rRNA)
TRANCRIPTION
transfer RNA (tRNA)

messenger RNA (mRNA)

Ribosomal RNA - constitutes about 80% of all RNA in E. coli cells. They are important structural
components of ribosome, which functions as non-specific sites of protein synthesis during translation.
Messenger RNA molecules- carry genetic information from the DNA of the gene. The mRNAs vary in size,
reflecting the range in the sizes of the proteins encoded by the mRNA.

Transfer RNA- accounts for up to 15% of the RNA in a typical cell. It carries amino acids to the ribosome
during translation, aiding in the translation of DNA to mRNA to protein.

Although ribosomal RNA and transfer RNA molecules are also synthesized by transcription of DNA
sequences, unlike mRNA molecules, these RNAs are not subsequently translated to form proteins, and
they remain in RNA form.

Other important RNA includes:

small nuclear RNA (snRNA), small nucleolar RNA (snoRNAs), micro RNA (miRNA), and small interfering
RNA (siRNA).

snRNA -participates in processing hnRNAs into mRNA and snoRNA primarily functions as RNA
chaperones in the processing of ribosomal RNA (rRNA). Both are neither tRNA nor small rRNA molecules,
although they are similar in size (100–200 nucleotides) to these species. Always found in a stable
complex with specific proteins forming small nuclear ribonucleoprotein particles (snRNP). Both miRNA
and siRNA are involved in gene regulation. The siRNAs disrupt gene expression by blocking specific
protein production hence their name “interfering,” even though the mRNA encoding the protein has
been synthesized.

miRNAs control developmental timing by base pairing with and preventing the translation of certain
mRNAs, thus, blocking synthesis of specific proteins.

unlike siRNAs, miRNAs (22 nucleotides long) do not cause mRNA degradation

 Transcription is the process in which a gene's DNA sequence is copied (transcribed) to make an RNA
molecule.
 RNA polymerases are enzymes that transcribe DNA into RNA.Using a DNA template, RNA polymerase
builds a new RNA molecule through base pairing. For instance, if there is a G in the DNA template, RNA
polymerase will add a C to the new, growing RNA strand.

RNA SYNTHESIS
Template Strand

Of the two strands of DNA, one of them is the template for RNA synthesis of a particular RNA product.
The enzyme that catalyzes the process is DNA-dependent RNA polymerase. RNA polymerase reads the
template from 3’ to 5’ (Fig. 3.1). All four ribonucleotides (ATP, GTP, CTP, and UTP) are required. The
strand that is used as template is called template strand, because it directs the synthesis of the RNA. It is
also called the antisense strand, because its code is the complement of the RNA that is produced. It is
sometimes called the strand by convention.

Coding Strand

The other strand is called the coding strand, because its sequence is the same as the RNA sequence that
is produced, with the exception of U replacing T. It is also called sense strand, because the RNA
sequence is the sequence that we use to determine what amino acids are produced through mRNA. It is
also called (+) strand, or nontemplate strand. As the RNA polymerase moves along the template strand
in 30 !50 direction, the RNA chain grows in 50 !30 direction. The nucleotide at the 50 end of the chain
retains its triphosphate group. Unlike DNA replication, a primer is not needed in RNA synthesis. The RNA
synthesized by the RNA polymerase is, therefore, named messenger RNA (mRNA) for its role in carrying
a copy of the genetic information to the ribosome.

THE PROCESS OF TRANSCRIPTION- usually broken down into three phases:

Initiation

There are usually four steps in the transcription initiation: (1) formation of a closed promoter complex,
(2) conversion of the closed promoter complex to an open promoter complex, (3) polymerizing the first
few nucleotides (up to 10) while the polymerase remains at the promoter, and (4) promoter clearance,
in which the transcript becomes long enough to form a stable hybrid with the template strand (Fig. 3.6).
This helps to stabilize the transcription complex, and the polymerase moves away from the promoter
and the transcription starts the elongation stage. Below is the detail of these four steps in the initiation
stage.
The initiation begins when RNA polymerase binds to the promoter and forms the closed complex. The σ
subunit directs the polymerase to the promoter by binding to specific sequences upstream of the start
site of transcription. Subsequently, both β’ and σ subunits initiate separation of the two downstream
DNA strands, melting about 10–17 bp surrounding the TSS, to form the open complex-transcription
bubble which is also known as the transcription bubble (Fig. 3.7). Next, the polymerase starts building
the RNA chain. The first base in RNA is a purine ribonucleoside triphosphate and A tends to occur more
often than G (Fig. 3.8). After the first nucleotide is in place, the polymerase joins a second nucleotide to
the first, forming the initial phosphodiester bond in the RNA chain. Both β and β’ subunits are involved
in phosphodiester bond formation as well as DNA binding. Several nucleotides may be joined before the
polymerase leaves the promoter and elongation begins. Within the transcription bubble are the nine
most recently added ribonucleotides of the RNA transcript
which remain base-paired to the template strand. The association of the polymerase with the RNA-DNA
hybrid within the transcription bubble contributes to the stability of the elongation complex. All cells
have a primary sigma factor which directs transcription from the promoters of gene encoding essential
proteins that are needed by growing cells. These genes are usually referred to as housekeeping genes.
On the other hand, bacteria also have a variety of alternative sigma factors. These alternative sigma
factors’ levels or activities regulated by the response to specific signals or stress conditions. Therefore,
these alternative sigma factors direct transcription of genes that are only required under certain
conditions. We will discuss the alternative sigma factor later.

Elongation

During the elongation phase, RNA polymerase directs the sequential binding of ribonucleotides to the
growing RNA chain in the 5’ to 3’ direction, while the RNA polymerase and transcription bubble move
along the template DNA in 30 !50 direction. As the RNA polymerase moves along the template DNA, the
transcription bubble also moves with it. This melted region exposes the bases of the template DNA one
by one so they can pair with the bases of the incoming ribonucleotides. When about 9-10 nucleotides
have been incorporated, the σ subunit dissociates from the holoenzyme and is later recycled to bind to
another core enzyme for another initiation process. The core enzyme continues to elongate the RNA,
adding one nucleotide after another to the growing RNA chain. The core subunit β lies near the active
site of the RNA polymerase where phosphodiester bonds are formed, and involves the bond formation.
As the RNA polymerase travels along the template DNA, the polymerase maintains a short melted region
of template DNA. This requires that the DNA unwind ahead of the advancing polymerase and close up
again behind it. During this process, positive supercoiling is produced ahead of the transcription bubble,
and negative supercoiling is produced behind the transcription bubble (Fig. 3.9). As such,
topoisomerases come in to relax the supercoils in front of and behind the advancing transcription
bubble. As soon as the transcription machinery passes, the two DNA strands wind around each other
again, reforming the double helix. The process of elongation is far from uniform and steady. Instead,
RNA polymerase frequently pauses, or even backtracks, while elongating an RNA chain. Pausing is
physiologically important for two reasons: first, it allows translation to keep pace with the RNA
polymerase. This is important for attenuation and aborting transcription if translation fails. Attenuation
is a mechanism to regulate the expression by causing premature termination of transcription of the
operon when the operon’s products are abundant. The second important aspect of pausing is that it is
the first step in transcription termination. The backtracking of RNA polymerase aids proofreading by
extruding the 30 -end of the RNA out of the polymerase, where misincorporated nucleotides can be
removed by an inherent nuclease activity of the polymerase, stimulated by auxiliary factors.

Termination

Termination of RNA transcription involves specific sequences downstream of the actual gene for the
RNA to be transcribed. There are two types of termination mechanism—intrinsic termination and rho (ρ)
—dependent termination. For
RNA Splicing

The mature transcript for many genes is encoded in a discontinuous manner in a series of discrete
exons. RNAs includes mRNAs, tRNA, and rRNA; all contain introns that must be removed from precursor
RNAs to produce functional molecules. tRNA precursors undergo splicing that is catalyzed by protein
factors and rRNA precursors catalyze their own removal and are termed self-splicing. On the other hand,
mRNA splicing is carried out by the spliceosome. Spliceosome is composed of several individual small
nuclear ribinucleoproteins (snRNP) and many more additional proteins that come and go during the
splicing reaction. snRNP removes the intervening sequences from pre-mRNAs, and they contain a large
number of proteins and five small nuclear RNAs (snRNAs), including U1, U2, U4, U5, and U6 snRNAs.
They are post-transcriptionally modified proteins. During the splicing, the 20 OH of an adenosine residue
located within the introns itself attacks the previous exon-intron boundary, detaching the intron from
the previous exon and producing a branched intron structure (Fig. 3.35). Next, the terminal 30 OH of the
newly released exon attacks the intron-next exon junction, splicing together the two exons and releasing
the intron. Before splicing can occur, the spliceosome must identify the splice sites between introns and
exons—the site at which exons are separated from their neighboring introns, and at which two exons
are subsequently attached. The spliceosome identifies splices sites by recognizing short sequence mot
NONCODING RNAs

-(ncRNA)comprised of 98% of transcriptional output of our genome The length of ncRNAs can vary from
21 to several thousand nucleotides (nt), and these molecules are divided into (i) long nc RNAs, such as
lncRNA, which are involved in epigenetic regulation of protein-coding gene expression and modulation
of gene transcription and protein degradation, and (ii) small ncRNAs, such as microRNAs (miRNAs), small
interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), and others.
Both miRNA and siRNA are involved in gene expression at the post-transcription stage. miRNA is
endogenous to the cell and produced by transcription of the cell’s gene while siRNAs are from
exogenous sources, such as a viral infection, or a synthetic dsRNA. Although miRNA and siRNA arise from
different sources, their mechanisms of action are similar.

The mRNA Degradation Mechanism Directed by siRNA

Dicer is one of the RNAase III endonucleases(enzyme that cuts phosphoester bond). It takes larger
dsRNAs and cut them to their characteristic small size leaving a two nucleotide-long 30 overhang. Once
the RNA is cleaved, it is passed to a protein complex RNA-induced silencing complex (RISC) (Fig. 3.37). In
an ATP-dependent process, RISC unwinds the double-stranded RNA and selects the antisense strand.
Subsequently, a nuclease protein in the RISC complex known as argonaut guides the complex to the
targeted mRNA. In the case of siRNA, the matching of the antisense strand to the mRNA is perfect. As
such, the argonaut protein cleaves the mRNA, and the mRNA is silenced. This is a process of RNA
interference.
The mRNA Degradation Mechanism Directed by miRNA

miRNAs are transcribed by RNA polymerase II from what are called MIR genes. The first intermediate
transcripts are “hairpin” molecules called pri-miR. Similar to mRNAs, these transcripts undergo capping
and polydenylation at the 5’ and 3’ ends of the transcript, respectively. Unlike mRNAs, miRNA
maturation begins in the nucleus and finishes in the cytoplasm. In animals, the nuclear processing is
performed by DROSHA, an RNase type III enzyme with endonucleolytic activity, which, in combination
with PASHA, recognizes the hairpin structure of the pri-miR and cleaves it. This generates a pre-miR of
approximately 60–70 nt in length. Pre-miRs are transported to the cytoplasm by exportin-5 and the
cofactor Ran-GTP. Once in the cytoplasm, pre-miRs are processed by the enzyme dicer. Dicer binds to
the pre-miRNA and cleaves it to miRNA. The miRNA then binds to RISC as it did with the siRNA. The
miRNA bound to the RISC structure is guided to the complementary sequence of the target mRNA. The
process is the same as RNA interference; if the pairing is complete (100% sequence complementarity
between the miRNA and mRNA), the mRNA will be degraded. If the miRNA is not a perfect match for the
mRNA target, there is no cleavage of the mRNA, but the RISC continues to bind to the mRNA, interfering
with the ability of ribosomes to translate mRNA. One unique aspect of miRNA regulation is its
complexity. It has been observed that a single miRNA can regulate expression of different mRNAs.
Additionally, one mRNA can be regulated by multiple miRNAs.

Both siRNA and miRNA Can Repress Transcription

In addition to repressing mRNA translation and triggering mRNA degradation, siRNA and miRNA can
also repress the transcription of specific genes and larger regions of the genome through associating
with a different complex—the RNA-induced initiation of transcription silencing complex (RITS). The
antisense RNA strand within the RITS targets the RITS complex to specific gene promoters or large
regions of chromatin. RITS then recruits chromatin remodeling enzymes to the promoters and these
enzymes methylate histones and DNA, resulting in heterochromatin formation and subsequent
transcriptional silencing (Fig. 3.38)
Amino Acids

- are building blocks of protein

General Structure of Amino Acids

includes an amino group, a carboxyl group, an α-carbon, a hydrogen, and an R group.

The R group, which is the side chain, determines the identity of the particular amino acid. Because the
α-carbon is located in the center with the other four different groups bonded to it, the α-carbon is the
chiral center in all amino acids except glycine, whose R group is hydrogen.
Chiral-symmetry /mirror image

Stereoisomers- molecules that have the same numbers of the same kinds of atoms and hence the
same formula but differ in chemical and physical properties.

a. Enantiomers-nonsuperimposable mirror-image stereoisomers

b. Diastereomer- nonsuperimposable nonmirror-image stereoisomers
A.

B.
Enantiomeric Molecules

-display a special property called optical activity: the ability to rotate the plane of polarization of plane-
polarized light. Clockwise rotation of incident light is referred to as dextrorotatory behavior, which
displays D-configuration, and counterclockwise rotation is called levorotatory behavior, which displays L-
configuration. The two possible stereoisomers of one chiral compound, L- and D-amino acids, represents
left and right form amino acids. The position of the amino group on the left or right side of the α-carbon
determines the L or D designation, respectively.

CLASSIFICATIONS OF AMINO ACIDS

There are four types of amino acids based on the features of the side chain: (1) nonpolar or hydrophobic
amino acids, (2) neutral (uncharged) but polar amino acids, (3) acidic amino acids, which have a net
negative charge at pH 7.0, and (4) basic amino acids, which have a net positive charge at pH 7.0.
Nonpolar Amino Acids

The nonpolar amino acids contain a nonpolar side chain.

Neutral (Uncharged) but Polar Amino Acids

The amino acids that have polar side chain are uncharged at neutral pH. The R group of polar uncharged
amino acids can form hydrogen bonds with water and play a variety of nucleophilic role in enzyme
reactions. These amino acids are usually more soluble in water than the nonpolar amino acids
Acidic Amino Acids That Have a Net Negative Charge at pH 7.0

Both glutamic acid and aspartic acid are acidic amino acids. They have carboxyl groups in their side
chains in addition to the one present in all amino acids. A carboxyl group can lose a proton, forming the
corresponding carboxylate anion. These forms are appropriately referred to as aspartate and glutamate.
Because of the presence of the carboxylate, the side chain of each of these two amino acids is negatively
charged at neutral pH.
Basic Amino Acids That Have a Net Positive Charge at pH 7.0

Histidine, lysine, and arginine have basic side chains, and the side chain in all three is positively charged
at the neutral pH. In lysine, the side-chain amino group is bonded to a hydrocarbon tail. In arginine, the
side-chain guanidino group is also bonded to a hydrocarbon tail. The histidine has an imidazole side
chain. The side chains of lysine and arginine are fully protonated at pH 7.0, but histidine is only partially
protonated at pH 7.0.