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RNA SYNTHESIS, PROCESSING &
MODIFICATION
BALDOMERO T. LASAM III RN MD
ASSOCIATE PROFESSOR 1
CSU-CMS
RNA
• A central tenet of molecular biology states that the flow of genetic
information in a cell is from DNA through RNA to proteins:
• DNA makes RNA makes Proteins
• RNA is the DNA photocopy
Biomedical Importance
Four Major Classes
• Ribosomal RNA
• Messenger RNA
• Transfer RNA
• Small RNA - involved in mRNA splicing and modulation of gene
expression by altering mRNA function
RNA is synthesized from a DNA Template by
an RNA Polymerase
• Process of DNA and RNA synthesis involve;
• 1. The general steps of initiation, elongation, and termination with 5'
to 3' polarity
• 2. Large, multicomponent initiation complexes
• 3. Adherence to Watson-Crick base paring rules
Difference of DNA and RNA Synthesis
• 1. Ribonucleotides are used in RNA synthesis rather than
deoxyribonucleotides
• 2. U replaces T as complementary base for A in RNA
• 3. Primer is not involved in RNA synthesis as RNA polymerases have
the ability to initiate synthesis de novo
• 4. Only portions of the genome are vigorously transcribed or
• copied into RNA, whereas the entire genome must be copied once
and only once during DNA replication
• 5. There is no highly active, efficient proofreading function during
RNA transcription
PURE AS GOLD!!!
• DNA consists of two types of bases, namely; purines and pyrimidines.
There are two types of purines: adenine and guanine, as well as two
types of Pyrimidines: cytosine and thymine. In the Watson-Crick DNA
base pairing model a purine always binds with a pyrimidine, however,
each purine binds to one particular type of pyrimidine.
• Adenine (A) binds to thymine (T) whilst, guanine (G) binds to cytosine
(C). However, in RNA uracil (U) is substituted for thymine (T). This
base pairing is referred to as complementary base pairing, hence the
base pairs are called complementary base pairs
Template Strand of DNA is Transcribed
• the sequence of ribonucleotides in an RNA molecule is
complementary to the sequence of deoxyribonucleotides in one
strand of the double-stranded DNA molecule
• Template strand - strand that is transcribed or copied into an RNA
molecule
• Non-Template strand - referred to as the coding strand of that gene
• The information in the template strand is read out in the 3' to 5'
direction
DNA Dependent RNA Polymerase Initiates
Transcription at a Distinct Site, the Promoter
• DNA Dependent RNA Polymerase is the enzyme responsible for the
polymerization of ribonucleotides into a sequence complementary to
the template strand of the gene
• The enzyme attaches at a specific site (promoter) on the template
strand
• Transcription Unit - defined as that region of DNA that includes the
signals for transcription initiation, elongation,and termination
• Primary Transcript - RNA product
Mammalian Cells Possess Three Distinct Nuclear
DNA Dependent RNA Polymerases
RNA Synthesis is a cyclical Process & involves RNA
Chain Initiation, Elongation & Termination
• Binding of the RNA polymerase haloenzyme molecule to the template at the
promoter site to form a Preinitiation complex or PIC
• Initiation of formation of the RNA molecule at its 5' end then follows, while
elongation of the RNA molecule from the 5' to its 3' end continues cyclically,
antiparallel to its template
• As the elongation complex containing the core RNA polymerase progresses
along the DNA molecule, DNA unwinding must occur in order to provide
access for the appropriate base pairing to the nucleotides of the coding strand
• Termination of the synthesis of the RNA molecule in bacteria is signaled by a
sequence in the template strand of the DNA molecule - a signal that is
recognized by a termination protein, the rho (p) factor
• Rho is an ATP-dependent RNA - stimulated helicase that disrupts the nascent
RNA-DNA complex
The Fidelity & Frequency of Transcription is
Controlled by Proteins Bound to Certain DNA
Sequences
Specific Signals Regulate Transcription Termination
Eukaryotic Transcription Complex
• a complex apparatus consisting of as many as 50 unique proteins
provides accurate and regulatable transcription of eukaryotic genes
• RNA polymerase enzymes (pol I, pol II and pol III) transcribe
information contained in the template strand of DNA into RNA
• These polymerases must recognize a specific site in the promoter in
order to initiate transcription at the proper nucleotide
• General Transcription Factors (GTFs) -facilitate promoter specific
binding of these enzymes and formation of the preinitiation complex
• Coactivators or coregulators - help regulate the rate of transcription
Promoter Accessibility and Hence PIC Formation is
Often Modulated by Nucleosomes
• on certain eukaryotic genes the transcription machinery (pol II,etc)
cannot access the promoter sequences (TATA-INR-DPE) because these
essential promoter elements are wrapped up in nucleosomes
• once the promoter is open following nucleosome eviction, pol II and
other essential proteins can bind and initiate mRNA gene
transcription
TATA BOX and Pribnow Box
• Pribnow box has a function similar to the TATA Box that occurs in
promoters in eukaryotes
• it is recognized and bound by a subunit of RNA polymerase during
initiation of transcription
• this region of the DNA is also the first place where base pairs separate
during prokaryotic transcription to allow access to the template
strand
Phosphorylation Activates Pol II
• Pol II is activated when phosphorylated on the Ser and Thr residues
and displays reduced activity when the CTD (carboxyl terminal repeat
domain) is dephosphorylated
• CTD phosphorylation/dephosphorylation is critical for promoter
clearance,elongation, termination and even appropriate mRNA
processing
• Pol II lacking the CTD tail is incapable of activation transcription and
cells expressing pol II lacking the CTD are inviable
The Role of Transcription Activators and
Coregulators
Two Models can Explain the Assembly of the
Preinitiation Complex
RNA Molecules are usually Processed before they
become functional
• Nearly all eukaryotic RNA primary transcripts undergo extensive
processing between the time they are synthesized and the time at
which they serve their ultimate function whether it be as
mRNA,miRNA's or as components of translation
• The process of transcription, RNA processing and even RNA transport
from the nucleus are highly coordinated
• Transcriptional coactivator termed SAGA in yeast and P/CAF in human
cells is thought to link transcription activation to RNA processing by
recruiting a second complex termed TREX
• TREX (transcription export) represents a likely molecular link between
transcription elongation complexes, the RNA machinery and nuclear
export
The Coding Portion (EXONS) of Most Eukaryotic
Genes are Interrupted by Introns
• one speculation for this exon-intron gene organization is that exons
which often encode an activity domain, or functional module of a
protein,represent a convenient means of shuffling genetic
information, permiting organisms to quickly test the results of
combining novel functional domains
Introns are Removed & Exons are Spliced Together
• Spliceosome - consist of the primary transcript, five small nuclear
RNA's (U1,U2,U4,U5 and U6) and more than 60 proteins many of
which containconserved RNP and SR protein motifs.
Alternative Splicing Provides for Different mRNA's
• the processing of mRNA molecules is a site for regulation of gene
expression
• Faulty splicing can cause disease
• B-thalassemia a disease in which the B-globin gene of hemoglobin is
severely underexpressed, appears to result from a nucleotide change
at an exon-intron junction, precluding removal of the intron and
therefore leading to diminished or absent synthesis of the B-chain
protein
Alternative Promoter Utilization Provides a Form
of Regulation
Both Ribosomal RNA's & Most Transfer RNAs are
Processed from Larger Precursors
RNA's Can Be Extensively Modified
• Mammalian mRNA molecules contain a 7-methylguanosine cap
structure at their terminal and most have a poly (A) tail at the 3'
terminal.
• The 5' cap of the RNA transcript is required both for efficient
translation initiation and protection of the 5' end of mRNA from
attack by 5'->3' exonucleases
• Poly (A) tail appears to protect the 3' end of mRNA from 3'-5'
exonuclease attack
Micro- RNA's are Derived from Large Pimary
Transcripts Through Specific Nucleolytic
Processing
RNA Editing Changes mRNA after Transcription
• the central dogma states that for a given gene and gene product
there is linear relationship between the coding sequence in DNA,the
mRNA sequence and protein sequence
Transfer RNA (tRNA) is Extensively Processed &
Modified
RNA can Act as a Catalyst
• Ribozymes
In Summary