LICYAYO, Michel Paulette

Chem 2065 AY2022-23

11 Transcription of the Genetic Code: The Biosynthesis of RNA

In this chapter, you are to learn about the process of converting the "information" stored
in DNA into RNA. in transcription, is the major control point in the expression of genes
and the production of proteins

In greater detail, you should be able to

1. Define the following terms: genome, chromosome, gene in the context of the
DNA structure
o Genome - the complete set of genetic instructions in an organism;
provides information that is needed to function
o Chromosome – long molecules of DNA found in the nucleus of the cell;
this is where the genome is stored
o Gene – made up of DNA; basic physical and functional unit of heredity
2. Provide an overview of the transcription process

Transcription is the process of turning the DNA into RNA and this is the major control
point in the expression of genes and the production of proteins. The process is carried
out by an enzyme called the RNA polymerase and accessory proteins called the
transcription factors. In transcription, the cells create a one stranded nucleic acid
molecule using a DNA template while following the rules of the complementary base
pairing. This single strand of RNA molecule carries the same information in one DNA
strand within a gene. This RNA molecule carries the genetic information to the
cytoplasm where the encoded information directs the formation of protein.

3. Differentiate between the template strand and the coding strand of DNA. Note
that these strands may have other names.
o Template strand – strand that directs the synthesis of the RNA; this is also
called the antisense strand or sometimes the (-) strand by convention
because its code is the complement of the produced RNA
o Coding strand – its DNA sequence will be the same as the produced RNA
sequence (except the U replacing the T); it’s also called as the sense
strand because the RNA sequence is used to determine what amino acids
are produced in the case of mRNA; also called the (+) strand by
convention or the nontemplate strand

4. Describe the properties of RNA Polymerase in E. coli (used as the

representative of prokaryotic systems)

RNA polymerase is the enzyme that synthesizes RNA and its molecular weight is
about 470,000 Da and has a multisubunit structure. There are five different types
of subunits identified as alpha, omega, beta, beta’, and sigma. The actual
composition of this enzyme is a2wBB’o. The sigma subunit is more loosely
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

bound to the rest of the enzyme, the a2wBB’ portion or the core enzyme and the
holoenzyme consists of all the subunits.

5. Differentiate between the functions of the RNA Polymerase holoenzyme and the
RNA Polymerase core enzyme

RNA polymerase is the enzyme that synthesizes RNA and its molecular weight is
about 470,000 Da and has a multisubunit structure. There are five different types
of subunits identified as alpha, omega, beta, beta’, and sigma. The actual
composition of this enzyme is a2wBB’o. The sigma subunit is more loosely
bound to the rest of the enzyme, the a2wBB’ portion or the core enzyme and the
holoenzyme consists of all the subunits.

6. Define the role of the sigma sub-unit in the binding of RNA Polymerase on the
DNA

The sigma-subunit of the RNA polymerase is involved in the recognition of

specific promoters. In bacteria, this subunit is involved in the initiation of
transcription which brings catalytically proficient RNA polymerase to promoters
through binding to specific DNA elements.

7. Define the role of the following components of the gene: promoter,

transcription start site (TSS), Pribnow box, -35 region aka -35 element, core
promoter, UP element, extended promoter
o Promoter – DNA sequences that provide direction for the RNA
polymerase; the portion of the DNA where the RNA polymerase binds at
the start of transcription
o Transcription Start Site (TSS) – (+1) position; the location on the DNA
template strand where the first ribonucleotide is used to initiate DNA
synthesis
o Pribnow box – DNA base sequence which is part of a prokaryotic
promoter and is located 10 bases before the transcription start site; (-10)
region
o -35 region aka -35 element – portion of the DNA that is 35 base pairs
upstream from the start of RNA transcription that is vital in control of the
RNA synthesis in bacteria
o Core promoter – the portion of the DNA from the transcription site to the -
35 region in prokaryotic transcription
o UP element – prokaryotic promoter element that is 40-60 bases upstream
of the TSS; enhances the binding of the RNA polymerase
o Extended promoter – region from the end of the UP element to the TSS*

8. Describe the chain initiation process by which RNA Polymerase binds to the
promoter region of the DNA
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

o Chain initiation starts with the recognition of the promoter by the sigma
subunit; binding of the holoenzyme to the DNA and the migration of the
promoter.
o Formation of the RNA polymerase, the closed promoter complex
o Unwinding of the DNA at the promoter and the formation of the open
promoter complex

9. Describe the chain elongation process by which RNA Polymerase elongates

the polynucleotide
o The RNA polymerase initiates the mRNA synthesis which is almost with a
purine
o The RNA polymerase holoenzyme-catalyzed elongation of mRNA by
about four more nucleotides
o The sigma subunit is released as the core RNA polymerase proceeds
down the template which elongates the RNA transcript

10. Describe the chain termination process by which transcription ends (intrinsic
termination, rho-dependent termination)
o Intrinsic termination – controlled by termination sites; involves formation of
hairpin loop in the RNA formed which stalls the RNA polymerase over a
region which is rich in adenine and uracil base pairs.
o Rho-dependent termination –also causes a hairpin loop to form but now
involves a special protein called rho. This binds to the RNA and moves
toward the transcription bubble and when this happens termination
happens.

11. Indicate why transcription is regulated in prokaryotes (alternative sigma

factors, enhances, operons, and transcription attenuation)

Transcription is regulated in prokaryotes because it is largely responsible for

controlling the level of protein production. Also, so that not all the RNAs and
proteins are produced in same quantities or at the same time. Transcription is
controlled in four ways:

o Alternative sigma factors – virus and bacteria

o Enhancers – DNA sequences that bind to a transcription factor and
increase the rate of transcription
o Operons – a group of operator, promoter, and structural genes
o Transcription attenuation – transcription is controlled after it has begun by
pausing the early release of incomplete RNA sequences
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

12. Describe how viruses and bacteria are able to regulate or control transcription

Viruses and bacteria are able to exert some control over which genes are
expressed by producing different sigma subunits that direct the RNA polymerase
to different genes.

13. Differentiate between enhancers and promoters

o Promoter – when DNA sequence is labeled as a promoter then the RNA
polymerase binds to that region.
o Enhancer – DNA sequence that is usually upstream of the promoter;
polymerase does not bind here; when these allow a response to changing
metabolic conditions, they are called as the response elements.

14. Describe how enhancers and silencers interact with transcription factors in
response to changing metabolic conditions in the cell

When binding the transcription factor increases the level of transcription, the
element is said to be an enhancer while if it decreases, the element is said to be
a silencer.

15. Use the lac operon to describe the process of controlling transcription through
operons (structural gene, operator (vs. promoter), inducer, repressor,
regulatory gene) [You can just skim over the other aspects]

The lac operon consists of structural genes that are vital in the metabolism of the
disaccharide lactose.

o lacZ gene – encodes the beta-galactosidase enzyme which is responsible

for catabolizing lactose sugar
o lacY gene – encodes a lactose permease which is responsible for
transportation of the lactose inside the cell
o lacA – encodes an enzyme with the transacetylase function

● Control sites for the gene expression are upstream of the structural genes
listed above. The control sites include both the promoter and operator
regions; RNA polymerase binds to the promoter

● Operator – DNA element where the repressor protein binds

● LacI gene – located upstream of the lac promoter or the Plac region. This
encodes the lac repressor which binds to the operator region as a
homotetramer

● Operator site is downstream of the lac promoter and the binding of the
repressor to the operator regions is to block the binding of RNA polymerase
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

16. Describe how transcription attenuation regulates transcription in prokaryotes

Prokaryotes have no separation of transcription and translation so the ribosomes

are attached to the mRNA while it is being transcribed. Trp operon is regulated
by transcription attenuation. The trp operon’s first gene is the trpL sequence
which codes for a leader peptide which contains two key tryptophan residues.
The translation of the mRNA leader sequence depends on the supply of the
tryptophan-charged tRNA. When it is high, transcription is terminated
prematurely – only after 140 nucleotides of leader sequence transcribed.
However, when it is low, the operon is translated normally.

17. Provide an overview of transcription in eukaryotes (major difference: three types

of RNA Polymerases)

Transcription in eukaryotes is more complex in eukaryotes compared to that of

prokaryotes. Three primary polymerases with different activities are known to
exist with each having different activities that are responsible for RNA synthesis.
Each one transcribes a different set of genes and recognizes a different set of
promoters:

o RNA Polymerase I – found in nucleolus and synthesizes precursors of

most ribosomal RNAs
o RNA Polymerase II – found in nucleoplasm and synthesizes mRNA
precursors
o RNA Polymerase III – found in nucleoplasm and synthesizers the tRNAs,
precursors of 5S ribosomal RNA. and a variety of other small RNA
molecules involved in mRNA processing and protein transport

18. Explain why RNA Polymerase II is the most extensively studied among the three
types of eukaryotic RNA Polymerases

The RNA Polymerase II is the most extensively studied because it is responsible

for the synthesis of mRNA from the protein-coding genes. It is the one
responsible for the protein synthesis and without the mRNAs it synthesizes, the
genetic code would not be used by the body. Early attempts of studying this
enzyme indicated that its activity is different from the prokaryotic RNA
polymerase.

19. Describe the features of the Pol II promoters (upstream elements, TATA box,
GC Box, CAAT box, Initiator element [Inr])
o Upstream elements – act as enhancers and silencers; In transcription, it is
the portion of the sequences closer to the 3’ end than the gene to be
transcribed, where the DNA is read from the 3’ to the 5’ end and the RNA
is formed from the 5’ to the 3’ end; in translation, it is nearer to the 5’ end
of the mRNA
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

o TATA box – in the -25 position (25 bases upstream of the TSS); has a
consensus sequence of TATAA(T/A)
o GC box – close to the core promoter; -40 position; has a consensus
sequence of GGGCGG
o CAAT box – close to the promoter extending to -110 position; has a
consensus sequence of GGCCAATCT
o Initiator element (lnr) – a loosely conserved sequence surrounding the
transcription start site in eukaryotic DNA

20. Describe the role of mediator in transcription activation and repression

Transcription activation requires a protein called the mediator. It serves to bridge

the promoter, RNA polymerase and general transcription machinery with specific
remote enhancers and silencers. The mediator bridges the promoter region with
the enhancer region to activate transcription or in the opposite case, it binds to
the silencer element, but fails to recruit RNA pol II to the promoter. The mediator
is also known to be the ultimate regulator of eukaryotic transcription essential to
the transcription of almost every RNA polymerase II-dependent gene. It is also
required for basal transcription of these genes.

21. Explain how the chromatin structure naturally represses transcription in

eukaryotes

Relief of responses is caused by the chromatin structure. This relief is dependent

on factors that is able to recognize the chromatin and alter the nucleosomes so
that the promoters may be accessible to the transcription machinery. The DNA is
wounded tightly on the nucleosomes and when it is tightly packed, the RNA Pol II
has no access to the promoter, gions, and transcription will not be able to occur.

22. Explain the roles of chromatin remodeling complexes and histone-modifing

enzymes in eukaryotic transcription (histone acetyltransferases, histone
deacetylase, histone code)
o Chromatin remodeling complexes – it mediates ATP-dependent
conformational changes in nucleosome structure that leads to transcription

o Histone-Modifying Enzymes – introduces covalent modifications into the

N-terminal tails of the histone core octamer
1. Histone acetyltransferases (HATs) – enzymes that acetylate lysine
residues on histone proteins
2. Histone deacetylase (HDAC) – enzyme that removes the acetyl
group from an acetylated lysine on a histone protein

23. Briefly explain the roles of the noncoding RNAs: miRNA, siRNA in RNA
interference and RNA silencing
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

o Micro RNA (miRNA) and small interfering RNA (siRNA) are involved in
control of gene expression through several related mechanisms. They are
bound to the RNA-induced silencing complex (RISC) and their antisense
strands serve to guide strands to the mRNA target. In siRNA, the guide
strand binding to the mRNA results in cleavage of the mRNA molecule
which prevents translation. On the other hand, in the case of the miRNA,
the binding is not a perfect match and there is no cleavage formed.
Instead, the RISC remains bound to the mRNA and prevents it from
participating in protein translation.
o RNA interference is where the small RNA molecules either bind to mRNA
causing its destruction so it cannot be translated or block its translation
directly; RNA silencing is a biological process where the RNA inhibits or
completely suppresses the transcription of a gene
24. Describe the structural motifs in DNA-binding proteins: helix-turn-helix, zinc
fingers, and basic-region leucine zipper

● Helix-turn-helix (HTH) – common motif found in the DNA-binding domain of

transcription factors; sequence of 20 amino acids which are relatively conserved
in many different DNA-binding proteins. The first helix is composed of the first 8
residues of the region and a sequence of 3 or 4 amino acids separates it from the
second helical region

● Zinc fingers – common motifs found in the DNA-binding region of transcription

factors; 12 amino acids looped out from the intersection of the two cysteines and
two histidine with the zinc ion. When the transcription factor of RNA Pol III binds
to the DNA, the repeated zinc fingers follow the major groove around the DNA

● Basic-region leucine zipper (bZIP) – common motif found in transcription

factors; half of the protein is composed of the basic region with many conserved
residues of lysine, arginine, and histidine. The second half contains a series of
leucine every seven residues. It is named zipper from the fact that the line of
hydrophobic residues interacts with a second analogous protein fragment via
hydrophobic bonds, interweaving themselves like a zipper.

25. Describe the common posttranscriptional RNA modifications in tRNA, rRNA

o tRNA – Trimming, addition of terminal sequences, and base modification

take place in the transformation of the initial transcript to mature tRNAs.
The trimming and addition of terminal nucleotides produce tRNAs with the
proper size and base sequence. Trimming of large precursors of
eukaryotic tRNAs take place in the nucleus, but most methylating
enzymes occur in the cytosol
o rRNA – processing of rRNAs is primarily a matter of methylation and of
trimming to the proper size.
LICYAYO, Michel Paulette
Chem 2065 AY2022-23

26. Describe the common posttranscriptional RNA modifications in mRNA

(capping, methylation, polyadenylate tail, exons, introns, split genes,
splicing reaction: lariats and snurps, alternate RNA splicing)
o Capping the 5’ end – protect against exonuclease degradation
o Polyadenylating at the 3’ end – protects mRNA from nucleases and
phosphates against degradation
o Splicing – process where non coding sequences of mRNA are removed
and the coding sequences are linked together
o The removal of the introns by splicing yields the correct mRNA and
introns are removed by specific splicing reactions.

27. Describe the catalytic role of RNA (ribozymes)

Ribozymes are RNA enzymes that catalyze chemical reaction. Its reactions are
similar to that of protein enzymes. They play a role in other vital reactions such
as the RNA splicing, transfer of RNA biosynthesis, and viral replication