Transcription of the

Genetic Code: The

Biosynthesis of RNA
Chapter 11, Biochemistry,
Campbell, Farrell,
McDougal
Chapter Outline
Transcription in eukaryotes
Posttranscriptional RNA modifications
Ribozymes

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TATA-Binding Protein (TBP) Binding to DNA
TFIID = TBP + TAFs
• contains ~17 protein subunits including
• TBP (TATA-binding protein)
• • Highly conserved across
eukaryotes
• • Binds to the minor groove
of the TATA box
– Saddle-shaped TBP lines
up with DNA
– Underside of the saddle
forces open the minor
groove
• – DNA at TATA box is bent
through ~80° curve
• TAFs (TBP-associated
factors) specific for class II
promoters

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• Important stages of transcription initiation by RNA
polymerase II:
• a) The TATA box is recognized by TATA-binding
protein (TBP). This initiates an assembly of many
general transcription factors (TFIIs) and RNA Pol II
• b) The DNA is unwound (closed pre-initiation
complex to open initiation complex)
• c) The first few nucleotides are transcribed, and the
CTD of Pol II is phosphorylated. This allows RNA Pol
II to “clear” the promoter and enter elongation.

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Elongation and Termination
• Elongation is controlled by:
• Pause sites, where RNA pol will hesitate
• Abortion or premature termination
• Antitermination, which proceeds past the normal
termination point
• Classes of elongation factors
• TFIIF, TFIIS, and positive-transcription elongation
factor (P-TEF) and negative-transcription elongation
factor (N-TEF)
• Termination begins by stopping RNA polymerase
• AAUAAA - Eukaryotic consensus sequence for
termination
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Regulation of Gene Expression
• Mediator: Giant protein
complex that bridges the
promoter and GTFs with
remote silencers + • contains
enhancers many
subunits:
• Enhancers + silencers
Med
• Regulatory sequences that proteins
augment or diminish
transcription, respectively
• DNA looping brings
enhancers into contact
with transcription factors
+ polymerase

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Posttranscriptional RNA Modifications
• tRNA, rRNA, mRNA are modified to give rise to
RNA's functional form
• Type of processing in prokaryotes can differ greatly
from that in eukaryotes, especially for mRNA
• Initial size of the RNA transcript is greater than the
final size???
Types of modifications (e.g.)
• Addition of terminal sequences (after transcription)
• Modification of the structure of specific bases, particularly in
tRNA

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Transfer RNA
• Precursor of several tRNAs is transcribed in one long
polynucleotide sequence
• Trimming, addition of terminal sequences, base
modification take place in the transformation of the
initial transcript to the mature tRNAs
• Usual types of base modification - Methylation and
substitution of sulfur for
oxygen

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Ribosomal RNA
• Processing of rRNA is primarily a matter of
methylation + of trimming to the proper size

• Base modifications in both prokaryotes + eukaryotes

are accomplished primarily by methylation

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Messenger RNA Processing
• 1) enzyme-catalyzed addition of 5’cap
• 2) splicing (intron removal) by spliceosome
• 3) enzyme-catalyzed addition of 3’ poly-A-tail

5 cap Poly(A) tail

5 3

5 untranslated Coding region 3 untranslated

region region

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Messenger RNA
• Cap at the 5′ end of the mRNA is a
guanylate residue that is methylated
at the N-7 position
• Modified guanylate residue is
attached to the neighboring residue
by a 5′-5′ triphosphate linkage
• 2′-OH group of the ribosyl portion of
the neighboring residue is frequently
methylated

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5’ cap

• to protect the 5’ end of the transcript from

exonucleases

• helps to allow the mature mRNA to be

exported to the cytoplasm

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Poly-A-tail
• String of A at the 3’ end of an mRNA
• Polyadenylate tail
• Added to the 3′ end before the mRNA leaves the
nucleus
• Protects the mRNA from nucleases, phosphatases

5 cap Poly(A) tail

5 3

5 untranslated Coding region 3 untranslated

region region

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Messenger RNA - Splicing
To make a functional mRNA, noncoding regions must be
removed.

Exons
are the coding regions of eukaryotic genes that will be part of the final
mRNA product.

Introns
the intervening noncoding sequence

Therefore, Eukaryotic genes are much larger than their

corresponding mature mRNA.

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Fig. 11.41 - The Organization of Split Genes in
Eukaryotes

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Splicing Reaction
• Removal of intervening sequences takes place in the nucleus
• Requires cleavage at the 5′ and 3′ end of introns and the
joining of the two ends
• Specific sequences make up the splice sites
• Branch site within the intron has a conserved sequence
• Depends on small nuclear ribonucleoproteins, or snRNPs, to
mediate the process
• When the exons are spliced together, a lariat forms in the
intron

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Events in spliceosome assembly

• For most eukaryotic

introns, their removal is
catalyzed by a nuclear
catalytic complex called
the spliceosome.
• The subunits of the
spliceosome are called
snRNPs (“snurps”) and
are made of both RNA
and protein (snRNP=
small nuclear
ribonucleoproteins).

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Alternative RNA Splicing
• Gene expression can be controlled at the level of
RNA splicing
• 5% of the proteins produced in humans have
isoforms based on alternative splicing
• Isoforms: Different forms of a protein produced by
alternative splicing reactions
• Regulatory proteins can affect recognition of splice
sites + direct alternative splicing

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 Alternative Splicing of Primary RNAs
• Introns are spliced out of primary RNA transcripts while it
is still in the nucleus
• Alternative splicing leads to production of different
mature mRNAs from the same primary transcript

• Introns can
be retained
• Exons can
be skipped

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Ribozymes
• Catalyze their own self-splicing
• Involved in protein synthesis

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