RNA Structure, Synthesis

, and
Processing
(Lecture # 4)
Transcription:
RNA synthesis, or transcription, is the
process of transcribing DNA nucleotide
sequence information into RNA
sequence information.

RNA synthesis is catalyzed by a large

enzyme called RNA polymerase.

Transcription produces:
• messenger RNAs (mRNAs)
• ribosomal RNAs (rRNAs)
• transfer RNAs (tRNAs)
• small RNA molecules

The basic biochemistry of RNA synthesis is

common to prokaryotes and eukaryotes, although
its regulation is more complex in eukaryotes.

RNA synthesis, like nearly all biological polymerization

reactions, takes place in three stages: initiation,
elongation, and termination.
Expression of genetic information
by transcription
 Ribosomal RNA
Protein synthesis

rRNAs make up about 80% of the total RNA in the cell

“S” is the Svedberg unit for sedimentation rate,

which is determined by the size and shape of the particle
Transfer RNA
tRNAs are the smallest
(4S) of the three major
types of RNA molecules.
tRNAs make up about
15% of the total RNA
in the cell.
High percentage of
unusual bases, for
example, dihydrouracil
and have extensive
intrachain base -pairing
to give characteristic
secondary and tertiary
structure.
Carries specific amino
acid, to the site of
protein synthesis where
it recognizes the genetic A. Characteristic transfer RNA (tRNA) B. Folded (tertiary) tRNA structure found in cells.
code sequence on an secondary structure (cloverleaf). D = D = dihydrouracil; Ψ = pseudouracil; T = thymine;
mRNA dihydrouracil; Ψ = pseudouracil; T = thymine; C = cytosine; A = adenine.
C = cytosine; A = adenine.
Messenger RNA
mRNA comprises only about 5% of the RNA in the cell

mRNA carries genetic information from DNA for use in protein

synthesis.

Polycistronic mRNA
mRNA carries information from more than one gene
Prokaryotes
Monocistronic mRNA
mRNA carries information from just one gene
Eukaryotes
TRANSCRIPTION OF PROKARYOTIC GENES
• Properties of prokaryotic RNA polymerase
• In bacteria, one species of RNA pol synthesizes all of the RNA
• RNA pol is a multisubunit enzyme that recognizes a nucleotide sequence (the
promoter region) at the beginning of a length of DNA that is to be transcribed.
• RNA is synthesized from its 5'-end to its 3'-end, antiparallel to its DNA template
strand
• Core enzyme: Five of the enzyme’s peptide subunits , 2α, 1b, 1b', and 1W, are
required for enzyme assembly (α, W) template binding (b'), and the 5'→3' RNA
polymerase activity (b), and are referred to as the core enzyme. However, this
enzyme lacks specificity (that is, it cannot recognize the promoter region on the DNA
template).
• Holoenzyme: The s subunit (“sigma factor”) enables RNA pol to recognize promoter
regions on the DNA. The s subunit plus the core enzyme make up the holoenzyme.
Steps in RNA synthesis
• Initiation, Elongation, and Termination
• A transcription unit extends from the promoter to the termination
region
• The initial product of transcription by RNA pol is termed the primary
transcript.
1. Initiation:
• Transcription begins with the binding of the RNA pol holoenzyme to a region
of the DNA known as the promoter, which is not transcribed.
• The prokaryotic promoter contains characteristic consensus sequences

–35 Sequence

Pribnow box
unwinding
transcription bubble
2. Elongation:
• The elongation phase is said to begin when the transcript (typically starting
with a purine) exceeds ten nucleotides in length.
• Sigma is then released, and the core enzyme is able to leave (“clear”) the
promoter and move along the template strand in a processive manner, serving
as its own sliding clamp.
• During transcription, a short DNA–RNA hybrid helix is formed.
• Like DNA pol, RNA pol uses nucleoside triphosphates as substrates and releases
pyrophosphate each time a nucleoside monophosphate is added to the
growing chain.
• Transcription is always in the 5'→3' direction.
• In contrast to DNA pol, RNA pol does not require a primer and does not appear
to have 3'→5' exonuclease (proofreading) activity.
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 r (rho) factor
Rho-independent termination
of
prokaryotic transcription.
A. DNA template sequence
generates a self-complementary
sequence in the nascent RNA.
B. Hairpin structure formed by the
RNA. “N” represents a
noncomplementary base; A =
adenine, T = thymine; G = guanine;
C = cytosine; U = uracil. [Note:
Termination of eukaryotic Termination Signal. A termination signal
transcription is not well understood.] found at the 3’ end of an mRNA
transcript consists of a series of bases
that form a stable stem-loop structure
and a series of U residues.
Rho-Dependent termination:
This requires the participation of an additional
protein, rho (r ), which is a hexameric ATPase with
helicase activity. Rho binds a C-rich “rho
recognition site” near the 5'-end of the nascent
RNA and, using its ATPase activity, moves along the
RNA until it reaches the RNA pol paused at the
termination site. The ATP-dependent helicase
activity of r separates the RNA–DNA hybrid helix,
causing the release of the RNA

Mechanism For the Termination of Transcription by r Protein.

This protein is an ATP-dependent helicase that binds the
nascent RNA chain and pulls it away from RNA polymerase and
the DNA template.
TRANSCRIPTION OF EUKARYOTIC GENES
• More complicated process than transcription in prokaryotes.
• Separate polymerases for the synthesis of rRNA, tRNA, and mRNA.
• Transcription factors (TFs) are involved.
• TFs bind to distinct sites on the DNA either within the core promoter region,
close (proximal) to it, or some distance away (distal).
• They are required both for the assembly of a transcription complex at the
promoter and the determination of which genes are to be transcribed.
• For TFs to recognize and bind to their specific DNA sequences, the chromatin
structure in that region must be altered (relaxed) to allow access to the DNA.
Chromatin structure and gene expression
Euchromatin-relaxed
Heterochromatin-condensed
Chromatin remodeling
Nuclear RNA polymerases of Eukaryotic cells
1. RNA polymerase I:
• This enzyme synthesizes the precursor of the 28S, 18S, and 5.8S rRNA in the
nucleolus.
2. RNA polymerase II:
• This enzyme synthesizes the nuclear precursors of mRNA that are
subsequently translated to produce proteins.
• RNA pol II also synthesizes certain small ncRNAs, such as
• snoRNA
• snRNA
• miRNA
• Promoters for RNA polymerase II:
• In some genes transcribed by RNA pol II, a sequence of nucleotides (TATAAA)
that is nearly identical to that of the Pribnow box is found centered about 25
nucleotides upstream of the transcription start site. This core promoter
consensus sequence is called the TATA, or Hogness, box.
• In the majority of genes, however, no TATA box is present. Instead, different
core promoter elements such as Inr (initiator) or DPE (downstream promoter
element) are present.
• The sequences serve as binding sites for proteins known as general
transcription factors (GTFs), which in turn interact with each other and with
RNA pol II
• General transcription factors :
• These are the minimal requirements for recognition of the promoter, recruitment
of RNA pol II to the promoter, and initiation of transcription at a basal level.
• GTFs are encoded by different genes, synthesized in the cytosol, and transit to
their sites of action, and so are trans-acting.
• In contrast to the prokaryotic holoenzyme, eukaryotic RNA pol II does not itself recognize and
bind the promoter. Instead, TFIID, a GTF containing TATA-binding protein and TATA-associated
factors , recognizes and binds the TATA box (and other core promoter elements ).
• TFIIF, another GTF, brings the polymerase to the promoter.
• The helicase activity of TFIIH melts the DNA, and its kinase activity phosphorylates
polymerase, allowing it to clear the promoter.
• Regulatory elements and transcriptional activators :
• Upstream of the core promoter are additional consensus sequences. Those close to the
core promoter (within 200 nucleotides) are the proximal regulatory elements, such as
the CAAT and GC boxes.
• Those farther away are the distal regulatory elements such as enhancers.
• Proteins known as transcriptional activators or specific trancription factors (STFs) bind
these regulatory elements. STFs bind to promoter proximal elements to regulate the
frequency of transcription initiation, and to distal elements to mediate the response to
signals such as hormones and regulate which genes are expressed at a given point in
time.
• A typical protein-coding eukaryotic gene has binding sites for many such factors.
• STFs have two binding domains. One is a DNA-binding domain, the other is a
transcription activation domain that recruits the GTFs to the core promoter as well as
“coactivator” proteins such as the HAT enzymes involved in chromatin modification.
A. Association of the general
transcription factors (TFIIs) and
RNA polymerase II (RNA pol II)
at the core promoters. [Note: The
Roman numeral II denotes the TFs
for RNA pol II.] B. Enhancer
stimulation of transcription.
CTF = CAAT box transcription
factor; Sp1 = specificity factor-1.
Role of enhancers in eukaryotic gene regulation:
• Enhancers are special DNA sequences that increase the rate of initiation
of transcription by RNA pol II.
• Enhancers are typically on the same chromosome as the gene whose
transcription they stimulate. However, they can
• 1) be located upstream (to the 5'-side) or downstream (to the 3'-side) of the
transcription start site
• 2) be close to or thousands of base pairs away from the promoter
• 3) occur on either strand of the DNA. Enhancers contain DNA sequences called
“response elements” that bind STFs (transcriptional activators).
• By bending or looping the DNA, these enhancer-binding proteins can
interact with other transcription factors bound to a promoter and with
RNA pol II, thereby stimulating transcription.
3. RNA polymerase III:
• This enzyme synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA.
• Mitochondrial RNA polymerase
• Mitochondria contain a single RNA pol that more closely resembles bacterial
RNA pol than the eukaryotic nuclear enzymes.
Post-transcriptional Modification of RNA
• A primary transcript is the initial, linear, RNA copy of a transcription
unit.
• The primary transcripts of both prokaryotic and eukaryotic tRNA and
rRNA are post-transcriptionally modified by cleavage of the original
transcripts by ribonucleases.
• tRNAs are then further modified to help give each species its unique
identity.
• In contrast, prokaryotic mRNA is generally identical to its primary
transcript, whereas eukaryotic mRNA is extensively modified both co-
and post-transcriptionally
5'→5' triphosphate linkage that is resistant to most nucleases
Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7-methyltransferase .
7-methylguanosine cap helps stabilize the mRNA and permits efficient initiation of translation.
40–250 adenine nucleotides attached to the 3'-end
Poly-A tail is added after transcription by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate.
Tail help stabilize the mRNA, facilitate its exit from the nucleus, and aid in translation. After the mRNA enters the cytosol,
the poly-A tail is gradually shortened.
 Splicing

• The process of removing introns and joining exons is called

splicing

• The molecular complex that accomplishes these tasks is

known as the spliceosome.

• Alternative splicing is a mechanism for producing a large, diverse

set of proteins from a limited set of genes

• Uracil-rich snRNAs form small nuclear ribonucleoprotein

particles (snRNPs, or “snurps,” designated as U1, U2, U4, U5,
and U6) mediate splicing.
Splicing Mechanism Used for mRNA Precursors