Lecture On Transcription and Translation

Transcription and Translation
How do genes encode

proteins?
Transcription animation
•https://www.youtube.com/watch?v=cXlv
21NCGxQ
•https://www.youtube.com/watch?v=vLz2
A1cjPH8
Table of Contents
• History: linking genes and proteins
• Getting from gene to protein: transcription
 Evidence for mRNA
 Overview of transcription
 RNA polymerase
 Stages of Transcription
•Promoter recognition
•Chain initiation
•Chain elongation
•Chain termination
 mRNA Synthesis/Processing
Table of Contents (continued)
•Getting from gene to protein: genetic
code
•Getting from gene to protein:
translation
 Translation Initiation
 Translation Elongation
 Translation Termination
History: linking genes and proteins
 Inborn
errors of metabolism: inherited human metabolic
diseases (more information)
•Genes are the inherited factors
•Enzymes are the biological molecules that drive
metabolic reactions
•Enzymes are proteins
•Question:
•How do the inherited factors, the genes, control the
structure and activity of enzymes (proteins)?
• Beadle and Tatum (1941) PNAS USA 27, 499–506.
• Hypothesis:
 If genes control structure and activity of metabolic
enzymes, then mutations in genes should disrupt
production of required nutrients, and that disruption
should be heritable.
• Method:
 Isolated ~2,000 strains from single irradiate spores
(Neurospora) that grew on rich but not minimal medium.
Examples: defects in B1, B6 synthesis.
• Conclusion:
 Genes govern the ability to synthesize amino acids,
purines and vitamins.
• 1950s: sickle-cell anemia
 Glu to Val change in hemoglobin
 Sequence of nucleotides in gene determines
sequence of amino acids in protein
 Single amino acid change can alter the function
of the protein
• Tryptophan synthase gene in E. coli
 Mutations resulted in single amino acid change
 Order of mutations in gene same as order of
affected amino acids
From gene to protein: transcription
•Gene sequence (DNA) recopied or
transcribed to RNA sequence
•Product of transcription is a
messenger molecule that delivers the
genetic instructions to the protein
synthesis machinery: messenger RNA
(mRNA)
Transcription: overview
•Transcription requires:
•ribonucleoside 5´ triphosphates:
 ATP, GTP, CTP and UTP
 bases are adenine, guanine, cytosine
and uracil
 sugar is ribose (not deoxyribose)
•DNA-dependent RNA polymerase
•Template (sense) DNA strand
•Features of transcription:
•RNA polymerase catalyzes sugar-
phosphate bond between 3´-OH of ribose
and the 5´-PO4.
•Order of bases in DNA template strand
determines order of bases in transcript.
•Nucleotides are added to the 3´-OH of the
growing chain.
•RNA synthesis does not require a primer.
•In prokaryotes transcription and
translation are coupled. Proteins are
synthesized directly from the primary
transcript as it is made.
•In eukaryotes transcription and
translation are separated.
Transcription occurs in the nucleus,
and translation occurs in the
cytoplasm on ribosomes.
Transcription: RNA Polymerase
• DNA-dependent
 DNA template, ribonucleoside 5´ triphosphates, and
Mg2+
• Synthesizes RNA in 5´ to 3´ direction
• E. coli RNA polymerase consists of 5 subunits
• Eukaryotes have three RNA polymerases
 RNA polymerase II is responsible for transcription of
protein-coding genes and some snRNA molecules
 RNA polymerase II has 12 subunits
 Requires accessory proteins (transcription factors)
 Does not require a primer
Stages of Transcription
•Promoter Recognition (formation of
closed, then open complex)
•Chain Initiation (incorporation of the
first few bases)
•Chain Elongation
•Chain Termination
Transcription: promoter recognition
• Transcription factors bind to promoter sequences and
recruit RNA polymerase.
• DNA is bound first in a closed complex. Then, RNA
polymerase denatures a 12–15 bp segment of the DNA
(open complex).
• The site where the first base is incorporated into the
transcription is numbered “+1” and is called the
transcription start site.
• Transcription factors that are required at every promoter
site for RNA polymerase interaction are called basal
transcription factors.
1. The closed complex of transcription (eukaryotic)
is formed in the following steps:
2. The TATA-binding protein (TBP) binds to the
TATA box.
3. TPB is bound by TFIIB, which also binds to the
DNA on either side of the TBP.
4. The TFIIB-TBP complex is bound by another
complex consisting of TFIIF and RNA pol II.
5. TFIIE and H bind to complete the closed
complex.
6. TFIIH has a helicase activity that can unwind the
DNA around the transcription start site (+1).
Control of transcription
Transcription start site usually a TATA box (not

always)
TBP (TATA-binding protein) binds, changing DNA

structure (Fig 6-18).
Recruits transcription factor II proteins (TFIIA,

B, …) then RNA Pol II
Collectively the transcription initiation complex

Promoter recognition: promoter sequences
•Promoter sequences vary considerably.
•RNA polymerase binds to different
promoters with different strengths; binding
strength relates to the level of gene
expression
•There are some common consensus
sequences for promoters:
 Example: E. coli –35 sequence (found 35
bases 5´ to the start of transcription)
 Example: E. coli TATA box (found 10
bases 5´ to the start of transcription)
Promoter recognition: enhancers
•Eukaryotic genes may also have
enhancers.
•Enhancers can be located at great
distances from the gene they regulate,
either 5´ or 3´ of the transcription start,
in introns or even on the noncoding
strand.
•One of the most common ways to
identify promoters and enhancers is to
use a reporter gene.
Promoter recognition: other players
•Many proteins can regulate gene
expression by modulating the strength of
interaction between the promoter and RNA
polymerase.
•Some proteins can activate transcription
(upregulate gene expression).
•Some proteins can inhibit transcription by
blocking polymerase activity.
•Some proteins can act both as repressors
and activators of transcription.
Transcription: chain initiation
•Chain initiation:
•RNA polymerase locally denatures the
DNA.
•The first base of the new RNA strand is
placed complementary to the +1 site.
•RNA polymerase does not require a primer.
•The first 8 or 9 bases of the transcript are
linked. Transcription factors are released,
and the polymerase leaves the promoter
region.
•Figure of bacterial transcription initiation.
Transcription: chain elongation
•Chain elongation:
•RNA polymerase moves along the
transcribed or template DNA strand.
•The new RNA molecule (primary
transcript) forms a short RNA-DNA
hybrid molecule with the DNA
template.
Transcription: chain termination
•Most known about bacterial chain
termination
•Termination is signaled by a sequence
that can form a hairpin loop.
•The polymerase and the new RNA
molecule are released upon formation
of the loop.
RNA processing
Newly synthesized transcripts (mRNA) are processed

(Fig. 6-21):
•Splice out intervening sequences (=introns) leaving

expressed sequences (exons)
•“Cap” 5’ end of RNA
•Poly-adenylate 3’ end (Poly A+ tail)

Transcription: mRNA synthesis/processing
• Prokaryotes: mRNA transcribed directly from DNA
template and used immediately in protein
synthesis
• Eukaryotes: primary transcript must be processed
to produce the mRNA
 Noncoding sequences (introns) are removed
 Coding sequences (exons) spliced together
 5´-methylguanosine cap added
 3´-polyadenosine tail added
• Removal of introns and splicing of exons can occur
several ways
 For introns within a nuclear transcript, a spliceosome is
required.
•Splicesomes protein and small nuclear RNA (snRNA)
•Specificity of splicing comes from the snRNA, some of
which contain sequences complementary to the splice
junctions between introns and exons
 Alternative splicing can produce different forms of a
protein from the same gene
 Mutations at the splice sites can cause disease
•Thalassemia • Breast cancer (BRCA 1)
•RNA splicing inside the nucleus on particles
called spliceosomes.
•Splicesomes are composed of proteins and
small RNA molecules (100–200 bp;
snRNA).
•Both proteins and RNA are required, but
some suggesting that RNA can catalyze the
splicing reaction.
•Self-splicing in Tetrahymena: the RNA
catalyzes its own splicing
•Catalytic RNA: ribozymes
From gene to protein: genetic code
• Central Dogma
 Information travels from DNA to RNA to Protein
•Is there a one-to-one correspondence
between DNA, RNA and Protein?
–DNA and RNA each have four nucleotides
that can form them; so yes, there is a one-
to-one correspondence between DNA and
RNA.
–Proteins can be composed of a potential 20
amino acids; only four RNA nucleotides: no
one-to-one correspondence.
–How then does RNA direct the order and
• How many bases are required for each amino
acid?
•(4 bases)2bases/aa = 16 amino acids—not
enough
•(4 bases)3bases/aa = 64 amino acid possibilities
• Minimum of 3 bases/aa required
• What is the nature of the code?
 Does it have punctuation? Is it overlapping?
 Crick, F.H. et al. (1961) Nature 192, 1227–32.
(http://profiles.nlm.nih.gov/SC/B/C/B/J/ )
 3-base, nonoverlapping code that is read from a
fixed point.
• Nirenberg and Matthaei: in vitro protein
translation
 Found that adding rRNA prolonged cell-free
protein synthesis
 Adding artificial RNA synthesized by
polynucleotide phosphorylase (no template,
UUUUUUUUU) stimulated protein synthesis
more
 The protein that came out of this reaction was
polyphenylalanine (UUU = Phe)
 Other artificial RNAs: AAA = Lys; CCC =Pro
• Nirenberg:
 Triplet binding assay: add triplet RNA,
ribosomes, binding factors, GTP, and
radiolabeled charged tRNA (figure)
•UUU trinucleotide binds to Phe-tRNA
•UGU trinucleotide binds to CYS-tRNA
 By fits and starts the triplet genetic code was
worked out.
 Each three-letter “word” (codon) specifies an
amino acid or directions to stop translation.
 The code is redundant or degenerate: more
than one way to encode an amino acid
•https://www.youtube.com/watch?v=Ikq
9AcBcohA
•https://www.youtube.com/watch?v=G2
yovIdpTVk
https://www.youtube.com/watch?v=2Bw
WavExcFI
From gene to protein: Translation
•Components required for translation:
 mRNA
 Ribosomes
 tRNA
 Aminoacyl tRNA synthetases
 Initiation, elongation and termination
factors
Translation: initiation
•Ribosome small subunit binds to mRNA
•Charged tRNA anticodon forms base pairs
with the mRNA codon
•Small subunit interacts with initiation factors
and special initiator tRNA that is charged
with methionine
•mRNA-small subunit-tRNA complex recruits
the large subunit
•Eukaryotic and prokaryotic initiation differ
slightly
Translation: initiation
•The large subunit of the ribosome contains
three binding sites
 Amino acyl (A site)
 Peptidyl (P site)
 Exit (E site)
•At initiation,
 The tRNAfMet occupies the P site
 A second, charged tRNA complementary
to the next codon binds the A site.
Translation: elongation
•Elongation
•Ribosome translocates by three bases after
peptide bond formed
•New charged tRNA aligns in the A site
•Peptide bond between amino acids in A and
P sites is formed
•Ribosome translocates by three more
bases
•The uncharged tRNA in the A site is moved
to the E site.
Translation: elongation
 EF-Tu recruits charged tRNA to A
site. Requires hydrolysis of GTP
 Peptidyl transferase catalyzes
peptide bond formation (bond
between aa and tRNA in the P site
converted to peptide bond between
the two amino acids)
 Peptide bond formation requires RNA
and may be a ribozyme-catalyzed
reaction
Translation: termination
• Termination
• Elongation proceeds until STOP codon reached
 UAA, UAG, UGA
• No tRNA normally exists that can form base
pairing with a STOP codon; recognized by a
release factor
• tRNA charged with last amino acid will remain at
P site
• Release factors cleave the amino acid from the
tRNA
• Ribosome subunits dissociate from each other

Lecture On Transcription and Translation

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Lecture On Transcription and Translation

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Transcription and Translation

How do genes encode

Transcription start site usually a TATA box (not

TBP (TATA-binding protein) binds, changing DNA

Recruits transcription factor II proteins (TFIIA,

Collectively the transcription initiation complex

Newly synthesized transcripts (mRNA) are processed

•Splice out intervening sequences (=introns) leaving

•“Cap” 5’ end of RNA

•Poly-adenylate 3’ end (Poly A+ tail)

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