BMS 202

Cell Biology
Dr. Tamer Salem
Biomedical Sciences

From DNA to Protein:

How Cells Read the Genome
 CHAPTER CONTENTS
• FROM DNA TO RNA

• FROM RNA TO PROTEIN

 Introduction

• Genetic information directs the synthesis of proteins.

FROM DNA TO RNA
 Portions of DNA Sequence Are Transcribed into RNA

• Many identical RNA copies can be made from the same gene
(produce many protein molecules as needed).

• Each gene can be

transcribed and translated
at different rate.
• RNA differ from DNA (nucleotides are ribonucleotides;
uracil (U) instead of thymine (T); single strand).
• Because RNA is a single-stranded molecule it can form
interamolecular base pairs and fold into specific
structures.

• The ability of RNA to fold into a complex 3D shape allows

it to carry out various functions in cells (conveying
information between DNA and protein; regulatory and
catalytic roles).
Folding following both
Folding following the conventional and
only the nonconventional
conventional base- base-pair interaction
pair interaction (A- (A-G & C-U)
U & C-G)
 Transcription Produces RNA That Is
Complementary to One Strand of DNA

• Transcription begins with opening and unwinding DNA

double helix.

• One of the strands will act as a template for RNA

synthesis. Transcription Produces RNA That Is
Complementary to One Strand of
DNA
Transcription differs from replications in different
aspects:

- The newly formed RNA does not hydrogen bonded to

the DNA.

- One strand of the dsDNA is transcribed.

- RNA is much shorter than DNA (transcribe certain

regions).
• The RNA polymerase moves stepwise along the DNA and
unwinds the DNA helix (5’ to 3’ direction).
• The rapid release of the RNA from the DNA strand allows
other RNA copies to be synthesized before the
completion of the first transcript.

• 1500 nucleotides require about 50 s to be transcribed.

• Overlapped transcription allows more than 1000 to be

synthesized in an hour.
RNA polymerase differs from DNA polymerase:

- Uses ribonucleotides

- Does not require a primer

- Not as accurate as DNA polymerase (1 mistake/104

nucleotides)
 Cells produce various types of RNA

• In eukaryotes, one gene / one mRNA.

• In bacteria, set of genes / one mRNA.

 Signals in DNA Tell RNA Polymerase Where to Start
and Finish Transcription
• Initiation of transcription is a critical process.

• RNA pol recognizes the start of a gene and bind firmly (differ
between bacteria and eukaryotes).

• In bacteria, RNA pol collides randomly and slides rapidly

looking for a promoter.

• Then the RNA pol opens up

the double helix immediately
in front of the promoter.

• Transcription continue until

encountering the terminator
(or stop site).
• In bacteria, a subunit of RNA pol (sigma factor) recognizes
the promoter sequence.

• Each base presents unique features to the outside of the

double helix.
• Which strands to be transcribed? (polarity of the promoter).

• Must use the 3’ to 5’ strand as a template.

• Not all genes are transcribed at the same direction of the DNA
as a whole.
 Initiation of Eukaryotic Gene Transcription Is
a Complex Process

Many of the principles for bacteria are applied to

eukaryotes except:

- Eukaryotic cells have three types of RNA pol (I, II an III).

- Require a large set of accessory proteins (transcription
factors).

- Mechanisms that control transcription initiation are much

more elaborate than in prokaryotes.

- Genes are spread throughout the genome.

- Transcription initiation deal with nucleosomes and

sophisticated chromatin structure that not present in
prokaryotes.
 Eukaryotic RNA Polymerase Requires General
Transcription Factors

• The general transcription factors assemble on the

promoter (position the RNA pol and pull apart the
DNA double helix).

• First, TFIID binds to TATA box (dramatic local

distortion).

• Used as a landmark, other TFs start to assemble

to form transcription initiation complex.

• TFIIH phosphorylates the

tail of the RNA pol to start.

• Only dephosphorylated
RNA poly can Initiate RNA
synthesis.
 Eukaryotic mRNAs Are Processed in the Nucleus

• In bacteria, ribosome starts translation before the completion

of RNA synthesis.

• In eukaryotes, RNA synthesized in the nucleus and translated

in the cytoplasm.

• RNA has to go through several

RNA processing steps before
transported to the cytosol
(capping, splicing, and
polyadenylation).
• Phosphorylation of the tail
of RNA polymerase II
allows RNA-processing
proteins to assemble there.

• The additional phosphates

are in addition to the ones
initiated the RNA synthesis.
• RNA capping: modify the 5’ end of the RNA transcript (methylated
guanine) (after the 25 nucleotides synthesized).

• Polyadenylation: the 3’end of the mRNA

initially trimmed by an enzyme to cut the
RNA at particular site and another enzyme
to add a series of repeated adenine (A)
nucleotides (few hundreds nucleotides
long).

• Both increase the stability of the RNA

molecule, facilitate its transport to the
cytoplasm, and mark it as mRNA.
 In Eukaryotes, Protein-Coding Genes Are Interrupted
by Noncoding Sequences Called Introns
• In bacteria, most proteins are encoded by uninterrupted DNA
sequence.

• In eukaryotes, most genes are interrupted by long and non

coding intervening sequences called introns.

• The scattered pieces of coding sequences are called exons or

expressed sequences.
• Most protein-coding human genes are broken into multiple
exons and introns.
 Introns Are Removed From Pre-mRNAs by RNA Splicing

• How does the cell determine which parts of the RNA

transcript to remove during splicing?

• Special sequences near each end of the intron.

A Adenine
G Guanine
C Cytosine
T Thymine
U Uracil
R Purine (A or G)
Y Pyrimidine (C or T)
N Any nucleotide
B Not A (G or C or T)
H Not G (A or C or T)
D Not C (A or G or T)
V Not T (A or G or C)
• Splicing machine cut out the intron
in the form of a “Lariat” structure.

• RNA splicing is carried out largely

by RNA molecules (snRNAs).

• snRNAs with additional proteins

form small nuclear
ribonucleoproteins (snRNPs).
• snRNPs (U1, U2, U4, U5, and U6) interact together to form
spliceosome.
• Importance of intron-exon type of gene arrangement:
- Alternative splicing (allows many different proteins to be
produced)- 95% of human genes thought to undergo
alternative splicing.
- Speed up the emergence of new proteins (mixing different
domains from different proteins).
- Housing important regulatory sequences such as miRNAs.
 Mature Eukaryotic mRNAs Are Exported from the
Nucleus
• Only small fraction (mature mRNAs) will be used.

• The remaining RNA fragments do not leave the nucleus.

• How does the cell distinguish between them?

• Transport mRNA is highly selective and based on (Cap-binding complex,

poly-A—binding proteins, proteins binding to the appropriately spliced
junction)- all participate in transportation.
• Waste RNAs are degraded and
their nucleotides are reused.
 mRNA Molecules Are Eventually Degraded in the
Cytosol

• The lifetime of the mRNA in the cytosol affect its protein

production (depending on the nucleotide sequence of the
mRNA and the type of cells).

• In bacteria mRNA lifetime is about 3 min.

• In Eukaryotes, mRNA lifetimes persist longer.

• These differences affected by 3’-untranslated region

(3’UTR).
The Earliest Cells May Have Had Introns in Their Genes

• The process of transcription is

universal, then why are RNA
transcripts handled differently in
eukaryotes and prokaryotes?

• Bacteria removed their introns to

reproduce faster or eukaryotes were
invaded with mobile genetic
elements and did not bother to
remove them.
FROM RNA TO PROTEIN
 The nucleotide sequence of an mRNA is translated into the
amino acid sequence of protein via the genetic code

• Codons are written from 5’-to-3’ direction.

• Codons for the same a.a. have the same nucleotides at the 1st
& 2nd positions and vary at the 3rd position.

• Most amino acids are represented by more than one codon.

• There are three possible reading frames.

• Only one encodes the actual message.

 tRNA Molecules Match Amino Acids to Codons in
mRNA
• The codons on an mRNA do not directly recognize their
corresponding amino acids.

• (~80 nucleotides) tRNAs do

• Two regions are crucial to the function of tRNAs:

- Anticodon
- A short single-stranded region at the 3’ end (a.a. attachment
site)

Ψ pseudouridine
D dihydrouridine
Both derived from U
• Some amino acids have more than one tRNA (different
anticodons).

• Some tRNAs can tolerate a mismatch at the third position

(wobble).

• Humans have nearly 500 different

tRNA genes, but only 48 anticodons are
represented among them.
Specific Enzymes Couple tRNAs to the Correct Amino Acid

• tRNAs charged with the corrected amino acid by aminoacyl-

tRNA synthetases.

• Different synthetase enzyme for each amino acid.

 The mRNA Message Is Decoded by Ribosomes

• Ribosome (ribosomal proteins; ribosomal RNAs

(rRNAs)
• The eukaryotic ribosome is a large complex of four rRNAs and
more than 80 small proteins
• Each ribosome has a binding site for mRNA and three
binding sites for tRNA (A, P, and E)

• One of the largest structure in the cell

• Two-thirds RNA and one-third proteins
• rRNAs and not the proteins are responsible for the ribosome’s
overall structure (proteins are to hold and stabilize the RNA
core)
 Translation takes place in a four-step cycle

1- A charged tRNA binds to A site

2- the carboxyl end of amino acid 3 is uncoupled and
form a peptide bond with amino acid 4
3- a shift of the large subunit (moves the two amino acid
3 and 4 to the E and P sites, respectively)
4- The small subunit moves three nucleotides
to restore its original position
 Specific Codons in mRNA Signal the Ribosome Where to
Start and to Stop Protein Synthesis

• In the test tube, ribosomes can translate any

RNA molecule.

• In a cell, a specific start signal is required to

initiate translation.

• Translation starts at AUG.

• First amino acid is methionine (usually

removed later by a specific protease).

• The initiator tRNA is distinct from the tRNA

that carries methionine.

• Only initiator tRNA can bind tightly to the P

site without the large subunit.
• In bacteria, there is no 5’ cap to start from.

• They contain specific ribosome-binding sequences (up to six

nucleotides) right upstream of the AUGs.

• In bacteria, ribosome can bind directly to a start codon if

preceded by the ribosome-binding site.

• A single mRNA can be polycistronic (encode several different

proteins).
End of translation:

• Stop codon (UAA, UAG, UGA)

• A release factor binds to the A site

when the ribosome reaches a stop
codon (alters the activity of the peptidyl
transferase by adding water instead).

• Ribosome is dissociated to start a

second translation process.
Proteins Are Made on Polyribosomes
• Also called polysomes

• Found in both bacteria and eukaryotes

• In bacteria translation is faster due to:

1) no RNA processing time
2) accessibility of mRNA to ribosome while
transcribing

Video
 Inhibitors of Prokaryotic Protein Synthesis Are Used
as Antibiotics

• Antibiotics take advantages of the subtle differences between bacteria

and eukaryotes in transcription and translation
 Controlled Protein Breakdown Helps Regulate the
Amount of Each Protein in a Cell

• Protein activity and longevity depend on:

- Rate of protein production
- Rate of degradation

• How does the cell control the protein lifetimes?

• Proteolysis (break down proteins

to their constituent amino acid).

• The eukaryotic cells use

proteasomes (in cytosol and
nucleus).

• It degrades short-lived and

misfolded proteins.
• A proteasome contains a central part (proteases with their
active sites face into the inner chamber) and two stoppers (a
large protein complex form of at least 10 types of protein
subunits) at each end.

• Protein stoppers bind to proteins destined to be degraded and

linearize them inside the chamber.

• Which proteins are selected for degradation?

- Marked proteins for degradation (ubiquitylated protein).
- Misfolded or damaged proteins expose a sequence buried in
the healthy proteins.
 There Are Many Steps Between DNA and Protein

• The final concentration of a

protein depends on the rate of
any of the many steps between
DNA to protein.

• Many proteins require

various modifications to
become fully functional
(such as phosphorylation
and glycosylation).

Video
 References

• Alberts et al., Essential Cell Biology, 4th edition.

 Cracking the genetic code

• Synthetic mRNAs added to the bacterial cell lysate containing

ribosomes, tRNAs, enzymes, and other small molecules.

• Radioactive amino acids are added to the mix (one type of

amino acid each time).
• Mixed polynucleotides gave mixed messages (UGU or
GUG; AGA or GAG) but useful information.

• Trinucleotide bound to
ribosomes and Phe-
tRNAs bound to the UUU
(trapping the triplets).