Transcription and translation lecture notes

© 2011 Pearson Education, Inc.
Lectures by Stephanie Scher Pandolfi
BIOLOGICAL SCIENCE
FOURTH EDITION
SCOTT FREEMAN
16
Transcription
and Translation
Review of previous chapter-
what are two linked genes? ; They are on the same chromosome
Start chapter lecture -
Transcription is the creation of a piece of RNA based on the information in
DNA .
What carries out translation? ; Ribosomes. Ribosomes read the mRNA
This will be the last chapter***
The main focus of this chapter-
•The details of transcription and translation.
•The differences between prokaryotes and eukaryotes

Key Concepts
After RNA polymerase binds DNA with the help of other
proteins, it catalyzes the production of an RNA molecule whose
base sequence is complementary to the base sequence of the DNA
template strand.
Eukaryotic genes contain regions called exons and regions called
introns; during RNA processing, the regions coded by introns are
removed, and the ends of the RNA receive a cap and tail.
Key points –
•mRNA in eukaryotes are modified in 3 ways
1.Cap added to the 5’ prime end
2.Poly A tail is added to the 3’ prime end
3.Splicing- Exons spliced/ together
*Reason- stabilize RNA to last
longer. Cap gives protection in
order for to stay longer
because they have to leave
nucleus before being
translated

Board drawing splicing

Key Concepts
Ribosomes translate mRNAs into proteins with the help of
intermediary molecules called transfer RNAs (tRNAs).
Each transfer RNA carries an amino acid corresponding to the
tRNA’s three-base-long anticodon.
In the ribosome, the tRNA anticodon binds to a three-base-long
mRNA codon, causing the amino acid carried by the transfer
RNA to be added to the growing protein.
The codons recruit each amino acid by means of tRNA
•There are 20 tRNAs
•They are anticodons that compliment the codons

Introduction
• A cell builds the proteins it needs from instructions encoded in its
genome according to the central dogma of molecular biology.

Overview of Transcription
• The first step in converting genetic information into proteins is
transcription, the synthesis of an mRNA version of the
instructions stored in DNA.
• RNA polymerase performs this synthesis by transcribing only one
strand of DNA, called the template strand.
• The other DNA strand is called the non-template, or coding
strand, which matches the sequence of the mRNA, except that
RNA has uracil (U) in place of thymine (T).

Characteristics of RNA Polymerase
Like the DNA polymerases, an RNA polymerase performs a
template-directed synthesis in the 5′ to 3′ direction. But unlike
DNA polymerases, RNA polymerases do not require a primer
to begin transcription. Because they string RNA together
• Bacteria have one RNA polymerase while eukaryotes have three
distinct types, RNA polymerase I, II, and III.

Initiation: How Does Transcription Begin?
• Initiation is the first phase of transcription.
– However, RNA polymerase cannot initiate transcription on its
own.
– Sigma, a protein subunit, must first bind to the polymerase.
In prokaryotic transcription is initiated when Sigma
recognizes special sequences in DNA helps RNA
polymerase where to start

What Role Does Sigma Play in Initiation?
• Sigma and RNA polymerase together form a holoenzyme, an
enzyme made up of a core enzyme and other required proteins.
• Prokaryotic RNA polymerase is a holoenzyme made up of the core
enzyme, which has the ability to synthesize RNA, and a sigma
subunit.
– Sigma acts as a regulatory factor, guiding RNA polymerase to
specific promoter sequences on the DNA template strand.
Promoter sequences in DNA direct RNA polymerase where
to transcribe gene

Bacterial Promoters
• Bacterial promoters are comprised of 40−50 base pairs and have
two key regions.
• The –10 box is found 10 bases upstream (in the opposite direction
of RNA polymerase movement during transcription) from the
transcription start site (the +1 site) and consists of the sequence
TATAAT.
• The –35 box, consisting of the sequence TTGACA, is 35 bases
upstream from the +1 site.
• All bacterial promoters have a –10 box and a –35 box, the
remainder of the promoter sequence varies.
***slide only used to explain

Eukaryotic Promoters
• Eukaryotes have a much more diverse and complex series of
promoters than do prokaryotes.
• Many of the eukaryotic promoters include a unique sequence called
the TATA box, centered about 30 base pairs upstream of the
transcription start site.
Skip slide

In Bacteria, Sigma Subunits Initiate Transcription
• Transcription begins when sigma, as part of the holoenzyme
complex, binds to the –35 and –10 boxes.
• Sigma, and not RNA polymerase, makes the initial contact with
DNA that starts transcription, supporting the hypothesis that sigma
is a regulatory protein.
• Most bacteria have several types of sigma proteins.
– Each type allows RNA polymerase to bind to a different type
of promoter and therefore a different kind of gene.
Skip slide

Transcription Initiation in Eukaryotes
• As with bacteria, the RNA polymerase does not bind directly to the
promoter.
• In eukaryotes, a group of proteins called basal transcription
factors bind to the DNA promoter, thus initiating transcription.
Need to know basal transcription factors***
• Basal transcription factors perform a similar function to bacterial
sigma proteins.

What Occurs Inside the Holoenzyme?
• Sigma opens the DNA double helix and the template strand is
threaded through the RNA polymerase active site.
• An incoming ribonucleoside triphosphate (NTP) pairs with a
complementary base on the DNA template strand, and RNA
polymerization begins.
• Sigma dissociates from the core enzyme once the initiation phase
of transcription is completed.
Skip slide

1. Where transcription
starts, in promoter
sequence from –10box to
-35box by polymerase
binding to sigma at the +1
site
2. Opens bubble in DNA to
string together NTPS
3. In Eukaryotes there is no
sigma instead
transcription factors
4. Here ribosomes can start
synthesizing mRNA as
soon as it is made be
because there is no nuclear
envelope 4
3
1
2

Elongation and Termination
• During the elongation phase of transcription, RNA polymerase
moves along the DNA template and synthesizes RNA in the 5' → 3'
direction. Polymerase continues down DNA to string together
nucleotides
• Transcription ends with a termination phase. In this phase, RNA
polymerase encounters a transcription termination signal in the
DNA template.
• In bacteria the transcription termination signal codes for RNA
forming a hairpin structure, which causes the RNA polymerase to
separate from the RNA transcript, ending transcription.

Transcription
Video Does not show the 3 modifications in
mRNA before leaving nucleus

RNA Synthesis
Skip slide

RNA Processing in Eukaryotes
• In bacteria, the information in DNA is converted to mRNA
directly. In eukaryotes, however, the product of transcription is an
immature primary transcript, or pre-mRNA. Before primary
transcripts can be translated, they have to be processed in a
complex series of steps.
Skip slide

The Discovery of Eukaryotic Genes in Pieces
• The protein-coding regions of eukaryotic genes are interrupted by
noncoding regions.
– To make a functional mRNA, these noncoding regions must be
removed.
• Exons are the coding regions of eukaryotic genes that will be part
of the final mRNA product. Exons stay
• The intervening noncoding sequences are called introns, and are
not in the final mRNA. Introns Taken out
• Eukaryotic genes are much larger than their corresponding mature
mRNA.

RNA Splicing
• The transcription of eukaryotic genes by RNA polymerase
generates a primary RNA transcript that contains exons and
introns.
– Introns are removed by splicing.
• Small nuclear ribonucleoproteins (snRNPs) form a complex
called a spliceosome. This spliceosome catalyzes the splicing
reaction.

Adding Caps and Tails to RNA Transcripts
• Primary RNA transcripts are also processed by the addition of a 5′
cap and a poly(A) tail.
With the addition of cap and tail and completion of splicing,
processing of the primary RNA transcript is complete. The
product is a mature mRNA.
• The 5' cap serves as a recognition signal for the translation
machinery.
• The poly(A) tail extends the life of an mRNA by protecting it from
degradation.
Above bullets review modification of
mRNA

Another example of cap and tail.

An Introduction to Translation
• In translation, the sequence of bases in the mRNA is converted to
an amino acid sequence in a protein.
• Ribosomes catalyze translation of the mRNA sequence into
protein.
Review of past information

Transcription and Translation in Bacteria
• In bacteria, transcription and translation can occur simultaneously.
Bacterial ribosomes begin translating an mRNA before RNA
polymerase has finished transcribing it.
– Multiple ribosomes attached to an mRNA form a
polyribosome. Poly: many
– Many ribosomes are simultaneously working on mRNA
– Description in next slide***
• In eukaryotes, transcription and translation are separated. mRNAs
are synthesized and processed in the nucleus and then transported
to the cytoplasm for translation by ribosomes.

What type of cell is this
Eukaryote or prokaryote ?
Prokaryote, because the ribosomes is able to attach to
mrna while being transcribed.
Nuclear envelope prevents this in Eukaryotes

Skip slide

Transcription and Translation in Eukaryotes
• In eukaryotes, transcription and translation are separated. mRNAs
are synthesized and processed in the nucleus and then transported
to the cytoplasm for translation by ribosomes.

How Does an mRNA Triplet Specify an Amino Acid?
• There were two hypotheses regarding the specification of amino
acid sequence by a sequence of nucleotide bases:
1. mRNA codons and amino acids interact directly.
2. Francis Crick proposed that an adapter molecule holds amino
acids in place while interacting directly and specifically with
a codon in mRNA.
• The adapter molecule was later found to be a small RNA called
transfer RNA (tRNA).

The Characteristics of Transfer RNA
• ATP is required to attach tRNA to an amino acid.
• Enzymes called aminoacyl tRNA synthetases “charge” the tRNA
by catalyzing the addition of amino acids to tRNAs.
• For each of the 20 amino acids, there is a different aminoacyl tRNA
synthetase and one or more tRNAs.
• A tRNA covalently linked to its corresponding amino acid is called
an aminoacyl tRNA.
Skip slide

What Happens to the Amino Acids Attached to tRNA?
• Experiments with radioactive amino acids revealed that they are
lost from tRNAs and incorporated into polypeptides synthesized in
ribosomes.
These results inspired the use of “transfer” in tRNA’s name,
because amino acids are transferred from the RNA to the growing
end of a new polypeptide. The experiment also confirmed that
aminoacyl tRNAs act as the interpreter in the translation process:
tRNAs are Crick’s adapter molecules.
Skip slide

What Do tRNAs Look Like?
• The CCA sequence at the 3' end of each tRNA is the binding site
for amino acids.
• The triplet on the loop at the opposite end is the anticodon that
base pairs with the mRNA codon. Important***
• The secondary structure of tRNA folds over to produce an L-
shaped tertiary structure.
All of the tRNAs in a cell have the same structure, shaped like an
upside-down L. They vary at the anticodon and attached amino
acid.

Reason why anticodons are
important, it lets the Ribosome
know that this codon should
bring this amino acid. The
amino acid is attached to this
particular tRNA that has
anticodon to compliment this
codon.

How Many tRNAs Are There?
• There are 61 different codons but only about 40 tRNAs in most
cells.
• To resolve this deficit, Francis Crick proposed the wobble
hypothesis. This hypothesis proposes that the anticodon of tRNAs
can still bind successfully to a codon whose third position requires
a nonstandard base pairing.
• Thus, one tRNA is able to base pair with more than one type of
codon.

The Structure and Function of Ribosomes
• Ribosomes contain protein and ribosomal RNA (rRNA).
• Ribosomes can be separated into two subunits:
– The small subunit, which holds the mRNA in place during
translation.
– The large subunit, where peptide bonds form.
• During translation, three distinct tRNAs line up within the
ribosome.

Ribosomes and the Mechanism of Translation
• All three tRNAs are bound at their anticodons to the corresponding
mRNA codon.
• There are 3 chambers inside the ribosome
– The A site of the ribosome is the acceptor site for an
aminoacyl tRNA. Acceptor of amino acid
– The P site is where a peptide bond forms that adds an amino
acid to the growing polypeptide chain. Polypeptide- where
they form
– The E site is where tRNAs no longer bound to an amino acid
exit the ribosome. Exit

Proccess will continue until
a stop codon is reached.
Stop codon recognized by
release factor protein. Slide
69***

Ribosomes and the Mechanism of Translation
The ribosome is a molecular machine that synthesizes proteins in
a three-step sequence.
1. An aminoacyl tRNA carrying the correct anticodon for the
mRNA codon enters the A site.
2. A peptide bond forms between the amino acid on the
aminoacyl tRNA in the A site and the growing polypeptide
on the tRNA in the P site.
3. The ribosome moves ahead three bases and all three tRNAs
move down one position; the tRNA in the E site exits.

The Phases of Translation
• Translation has three phases: initiation, elongation, and
termination.

Initiation
• The initiation phase of translation begins at the AUG start codon.
• ***there are many AUG codons
• In bacteria, the start codon is preceded by a ribosome binding site
(also called the Shine-Dalgarno sequence) that is complementary
to a section of one rRNA in the small ribosomal subunit. The
Shine-Dalgarno sequence follows the proper start codon in for
the ribosome to know where to start in Prokaryotes cells
• Eukaryotes use a sequence called a kozak sequence***
• The interaction between the small subunit and the mRNA is
mediated by initiation factors.

Initiation in Bacteria
• Translation initiation is a three-step process in bacteria:
1. The mRNA binds to a small ribosomal subunit.
2. The initiator aminoacyl tRNA bearing N-formylmethionine
(f-met) binds to the start codon.
3. The large ribosomal subunit binds, completing the complex.
• Translation is now ready to begin.

Elongation
• At the start of the elongation phase, the initiator tRNA is in the P
site, and the E and A sites are empty.
• An aminoacyl tRNA binds to the codon in the A site via
complementary base pairing between anticodon and codon.
• Peptide bonds form between amino acids on the tRNAs in the P and
A sites.
– After peptide bond formation, the polypeptide on the tRNA in
the P site is transferred to the tRNA in the A site.

Is the Ribosome an Enzyme or a Ribozyme?
• The active site of the ribosome is entirely ribosomal RNA.
• Thus, ribosomal RNA catalyzes peptide bond formation and the
ribosome is a ribozyme.

Moving Down the mRNA
• Translocation occurs when elongation factors move the mRNA
down the ribosome three nucleotides at a time, and the tRNA
attached to the growing protein moves into the P site.
• The A site is now available to accept a new aminoacyl tRNA for
binding to the next codon.
• The tRNA that was in the P site moves to the E site, and if the E
site is occupied, that tRNA is ejected.

Elongation
• Elongation has three steps:
1. Arrival of the aminoacyl tRNA.
2. Peptide bond formation.
3. Translocation.

Termination
• The termination phase starts when the A site encounters a stop
codon.
• This causes a protein called a release factor to enter the site.
Release factors resemble tRNAs in size and shape but do not carry
an amino acid.
• These factors catalyze hydrolysis of the bond linking the tRNA in
the P site with the polypeptide chain.
• Proccess will continue until a stop codon is reached.
• Stop codon recognized by release factor protein. Slide 69***

Release factor has no anticodon.
It is a protein causes everything to be released
(like a bomb) and stopping translation

Translation
Skip slide

Synthesizing Proteins
Video viewed in class
lecture

Post-Translational Modifications
• Most proteins go through an extensive series of processing steps,
collectively called post-translational modification, before they
are ready to go to work in a cell.
• Molecular chaperones speed folding of the protein. Folding
determines a protein's shape and therefore its function. From
previous chapters we learned how after translation chaperones
and folding are important.
• Many proteins are altered by enzymes that add or remove a
phosphate group. These changes often switch the protein from an
inactive state to an active state or vice versa.

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