1. Transcription and Translation
BIOL 105
Dr. Corl
October 25, 2013

2. The Central
Dogma
• DNA codes for
RNA, which codes
for protein.

3. The Central Dogma
• Transcription is a process in which:
– The sequence of bases in a particular stretch of
DNA (a gene) specifies the sequence of bases in
an mRNA molecule.
• Translation is a process in which:
– A particular mRNA molecule then specifies the
exact sequence of amino acids in a protein.
• Thus, genes ultimately code for proteins.

4. Transcription in Bacteria
• In transcription:
– Instructions stored in DNA are “transcribed”
through the synthesis of an mRNA transcript.
• Transcription performed by RNA polymerase.
• Three phases of transcription:
– Initiation, elongation, and termination.

5. Transcription in Bacteria
• mRNA transcript is synthesized by RNA polymerase
in the 5’ to 3’ direction.
• Template strand:
– DNA strand that is read (transcribed) by RNA polymerase.
• Non-template strand:
– DNA strand that is not read by RNA polymerase.

6. Transcription in Bacteria
• “Activated” complementary ribonucleotide
monomers are added on via condensation
reactions, resulting in phosphodiester bonds.

7. Bacterial RNA Polymerase
• A holoenzyme:
– An enzyme made up of a core enzyme and other
required proteins.
– Bacterial RNA polymerase is made up of a core
enzyme and a regulatory subunit, sigma.

8. Bacterial RNA Polymerase
• Core enzyme:
– Contains the active site where mRNA is synthesized.
• Sigma:
– A regulatory factor required for initiation of transcription.
– Tells core enzyme where in a DNA sequence to start transcription.

9. Transcription: Initiation
• Transcription initiation begins at specific
sequences of DNA called promoters.
• Two important bacterial promoter regions :
– Named the -10 box and the -35 box.
– Sigma recognizes these promoter regions and
brings the RNA polymerase core enzyme to the
promoter to initiate transcription.

10. Transcription: Initiation
Core enzyme
• 1.) sigma binds to specific promoter
regions of DNA (-35 box and -10 box).

11. Transcription: Initiation
• 2.) DNA double helix is opened and the
template strand of DNA is threaded through
RNA polymerase core enzyme active site.
mRNA synthesis begins.

12. Transcription: Initiation
• 3.) Initiation is complete.
Sigma dissociates from core enzyme.
mRNA synthesis (transcription) continues.

13. Transcription: Elongation
• Core enzymes of RNA polymerase moves along
the DNA template and continues to catalyze the
addition of complementary ribonucleotides to the
growing mRNA transcript.

14. Transcription: Termination
• RNA polymerase
encounters a
termination signal
within the DNA
template, which
codes for RNA
forming a hairpin
loop structure.
• Hairpin causes RNA
polymerase to
separate from RNA
transcript, ending
transcription.

15. Bacterial Transcription: Summary
• Initiation:
– Sigma brings RNA polymerase holoenzyme to
promoter region of DNA.
– DNA helix is opened and transcription begins.
– Sigma releases and transcription continues.
• Elongation:
– Complementary ribonucleotides are added to the
growing mRNA transcript as specified by the DNA
template strand.
• Termination:
– RNA polymerase reaches a termination signal in
the DNA template.
– mRNA forms a hairpin loop.
– mRNA dissociates from RNA polymerase.

16. Transcription in Eukaryotes
• Overall similar to bacterial transcription.
• Some differences include:
– Basal transcription factors:
• Proteins that bind DNA promoters independant of
RNA polymerase.
• RNA polymerase then binds to basal transcription
factors and transcription begins.
– Greater diversity and complexity of promoters:
• Many promoters include a TATA box sequence.

17. Transcription in Eukaryotes
• Three types of RNA polymerase:
– RNA polymerase II:
• Catalyzes transcription of genes that code for
proteins, forming mRNA.
– RNA polymerase I and RNA polymerase III:
• Catalyze transcription of non-protein coding
genes (e.g. genes coding for ribosomal rRNAs
and genes coding for transfer tRNAs).

18. Eukaryotic mRNA Processing
Exon 1
Intron 1
Intron 2
Exon 2
Exon 3
• After transcription but before translation, specific
regions of the primary RNA transcript are spliced
out (cut out) and degraded during RNA processing.
• The term intron can be used in two ways:
– A stretch of RNA that does get spliced out and will NOT
be a part of the final mature spliced RNA transcript.
– A stretch of DNA that codes for an RNA intron.

19. Eukaryotic mRNA Processing
Exon 1
Intron 1
Intron 2
Exon 2
Exon 3
• After transcription but before translation, specific
regions of the primary RNA transcript are spliced
out (cut out) and degraded during RNA processing.
• The term exon can be used in two ways:
– A stretch of RNA that does NOT get spliced out and will
be a part of the final mature spliced RNA transcript.
– A stretch of DNA that codes for an RNA exon.

20. Eukaryotic mRNA Processing
-Intron regions of the primary mRNA transcript are
spliced out by small nuclear ribonucleoproteins
(snRNPs), which assemble to form a spliceosome
(a ribozyme).

21. Eukaryotic mRNA Processing
• Intron regions of the primary mRNA transcript
are spliced out by snRNPs, which assemble to
form a spliceosome. (occurs within nucleus)

22. Eukaryotic mRNA Processing
• Additionally, a 5’ cap is added to the
very 5’ end of the mRNA transcript:
– 5’ cap serves as recognition signal for the
translation machinery of the cell.

23. Eukaryotic mRNA Processing
• Finally, a poly (A) tail is added to the
very 3’ end of the mRNA transcript:
– Extends the life of the mRNA by protecting
it from enzymatic degradation.

24. Transcription: Summary
• Note the similarities and differences between
bacterial vs. eukaryotic transcription!
• Eukaryotic transcription tends to be more complex.

25. Translation
• In translation:
– The sequence of bases in mRNA is
converted (“translated”) to an amino acid
sequence of a protein.
• Ribosomes catalyze the translation of
mRNA sequence into protein.

26. Translation in Bacteria
• In bacteria, transcription and translation can
occur simultaneously:
– Ribosomes begin translating an mRNA even before
RNA polymerase has finished synthesizing it!

27. Translation in Bacteria
• In bacteria, transcription and translation can
occur simultaneously:
– Ribosomes begin translating an mRNA even before
RNA polymerase has finished synthesizing it!

28. Translation in
Eukaryotes
• Transcription and translation are separated:
– Transcription takes place in the nucleus.
– Translation takes place in the cytoplasm.

29. Translation
• How do mRNA codons interact with amino acids?

30. Translation
• Adapter molecules:
– Small RNAs called transfer RNA (tRNA)
– Hold amino acids in place and interact directly and
specifically with a codon in mRNA.

31. Transfer RNA (tRNA)
• Different tRNAs covalently link to
specific amino acids, forming aminoacyl
tRNAs.
• Aminoacyl tRNA synthesizes:
– Enzymes which catalyze the addition of
amino acids to tRNAs.

32. Loading Amino Acid Onto tRNA
Step 1
Step 3
Step 2
Step 4
• Amino acid binds to its specific aminoacyl tRNA
sythnetase, which then covelantly attaches the
amino acid onto a specific tRNA.

33. Loading Amino Acid Onto tRNA
Step 1
Step 3
Step 2
Step 4
• Is ATP expended in this process? Yes!
• Aminoacyl tRNA = “CHARGED” tRNA

34. Aminoacyl tRNA
• Aminoacyl tRNAs then travel to ribosomes and
transfer amino acids to growing polypeptides.
• Each tRNA carries a specific amino acid that can
be transferred to protein.

35. Transfer RNA Structure
• tRNA secondary
structure resembles a
“cloverleaf.”

36. tRNA Secondary Structure
• 3’ end of tRNA: binding site for amino acids.
• Triplet loop at opposite end (anticodon):
– Interacts with complementary codon on mRNA.

37. tRNA Tertiary Structure
• tRNA has an “L-shaped” tertiary structure.
• Each tRNA has a distinct anticodon and attached
amino acid.

38. Wobble Hypothesis
• There are 61 different codons but only
40 different tRNAs.
• Explained by “wobble hypothesis”:
– The anticodon of certain tRNAs can bind
successfully to a codon whose third
position requires nonstandard base pairing.
– Allows one tRNA to be able to base pair
with more than one type of codon.

39. Ribosomes
• Composed of both ribosomal protein and
ribosomal RNA (rRNA).
• Can be separated into two subunits:
– Large subunit and small subunit.
• There are three sites within a ribosome that
tRNAs can reside:
– The A site, the P site, and the E site.

40. Ribosome: Structure
Large subunit
E
P
A
Small subunit
• Large subunit and small subunit.
• Three sites where tRNAs can reside: A, P and E.

41. Ribosome: Structure and Function
• Each tRNA binds at its anticodon to its corresponding
mRNA codon and transfers its amino acid to a
growing polypeptide chain.

42. Phases of Protein Synthesis
• initiation:
– Ribosomal subunits and mRNA assemble.
– Translation begins at the AUG start codon.
• elongation:
– Amino acids are transferred one by one from
aminoacyl tRNAs to a growing polypeptide chain.
• termination:
– Stop codon in mRNA causes protein to be
released from ribosome.
– Ribosomal subunits separate from mRNA and
from one another.

43. Initiation: Step 1
• mRNA is targeted to the ribosome:
– Ribosome binding site on mRNA binds to
complementary sequence on small subunit of ribosome,
with the help of proteins called initiation factors.

44. Initiation: Step 2
• Translation begins at the AUG start codon:
– Initiator aminoacyl tRNA, bringing in the first amino acid,
binds to the mRNA’s start codon, AUG.

45. Initiation: Step 3
• LARGE subunit of ribosome binds, placing initiator
aminoacyl tRNA in the P-site.

46. Elongation: Step 1
• Incoming aminoacyl tRNA binds to the codon in
theA site via complementary base pairing
between anticodon and codon.

47. Elongation: Step 2
• The amino acid in the P site:
– Breaks the bond with its tRNA.
– Forms a peptide bond with the amino acid in the A site.

48. Elongation: Step 3
• Translocation:
– Ribosome moves down to the next codon on mRNA.
– Results in tRNAs being shifted over one spot:
• tRNA in P site moves to E site.
• tRNA in A site moves to P site.

49. Another Elongation Cycle
• 1.) Incoming aminoacyl tRNA enters into the __ site.
• 2.) peptide bond formation.
– rRNA acts as a catalyst: ribozyme
• 3.) Translocation.

50. Polyribosome
• multiple ribosomes assembled along a single
mRNA, synthesizing proteins from the same
mRNA at the same time.

51. Termination: Step 1
• Occurs when a stop codon is revealed in the A site.
• A protein called a release factor enters the A site
and promotes the hydrolysis of the bond linking the
tRNA in the P-site with its polypeptide.

52. Termination: Steps 2 and 3
• Hydrolysis reaction (cleavage of bond between
tRNA and polypeptide) frees the polypeptide
• Ribosomal subunits, mRNA, tRNAs, and
polypeptide all _________ from one another.

53. Post-Translational Modifications
• Many proteins are modified after
translation is complete:
– Many proteins fold with the help of
molecular chaperones.
– Proteins can be modified by attachment of
sugars (glycosylation) in the rough ER
and/or Golgi.
– Proteins can be phosphorylated or
dephosphorylated, modifying their activity.

54. Mutations
• Mutation:
– Any permanent change in an organism’s DNA.
– Changes the genotype (DNA) of the cell.
• This may then result in a change in mRNA transcript
sequence, resulting in:
– Change in sequence of amino acids of the translated protein.

55. Point Mutations
• A single base pair change, often as a
result of errors in DNA replication.
• Can affect the primary structure of the
polypeptide that is ultimately translated.

56. Point Mutations
• missense mutation:
– Point mutation in a gene’s DNA sequence that
ultimately results in a single amino acid change in the
protein encoded by that gene.
– Can often be deleterious:
• Reduces the individual’s fitness.

57. Point Mutations
• silent mutation:
– Does not change amino acid sequence.
• nonsense mutation:
– Results in an early stop codon: shortened protein.
• frameshift mutation:
– Addition or deletion of a nucleotide causes entire reading
frame to be shifted.

58. Chromosome-Level Mutations
• Chromosome INVERSION and translocation:
– Can result in altered patterns of gene expression.

59. Summary
• Transcription:
– Bacterial transcription
– Eukaryotic transcription
• Translation:
– Ribosomes
– tRNA and aminoacyl tRNA
– Ribosomal protein synthesis
• Mutations:
– Point mutations
– Chromosomal mutations

60. Review Questions on
Transcription
• Contrast the functions of sigma versus the
core enzyme of bacterial RNA polymerase.
• What events occur during transcription
initiation? Elongation? Termination?
• How does eukaryotic transcription differ from
bacterial transcription?
• What types of post-transcriptional processing
occur in eukaryotes? What is splicing?

61. Review Questions on
Translation
• What is the function of tRNA? mRNA? rRNA?
• What events occur during the three major phases of
translation?
• What are the A, P, and E sites of a ribosome?
• What are the differences between silent, missense,
nonsense, and frameshift mutations?