1. RNA and Protein Synthesis

2. How does DNA “code”? DNA inherited by an organism
Leads to specific traits by dictating synthesis of proteins
DNA directs protein synthesis/gene expression
2 stages - transcription and translation

3. Central Dogma
DNA  RNA  PROTEIN

5. History of RNA Synthesis
George Beadle & Edward Tatum in (‘40s)- ‘One gene, one enzyme’
Function of a gene is to dictate the production of a specific enzyme
Beadle
American
Nobel Prize 1958
Tatum
American
Nobel Prize 1958

6. After Beadle & Tatum Some genes encode proteins that are not enzymes
One gene is responsible for one polypeptide chain, and some proteins have more than one chain
One gene, one polypeptide hypothesis

7. The BIG Picture DNA is transcribed to form RNA
RNA is translated to form protein

8. TRANSLATION
TRANSCRIPTION
DNA
mRNA
Ribosome
Polypeptide
Nuclear
envelope
TRANSCRIPTION
DNA
Pre-mRNA
RNA PROCESSING
mRNA
Ribosome
Prokaryotic cell - no nucleus, mRNA produced by transcription immediately translated without additional processing
TRANSLATION
Polypeptide
Eukaryotic cell - nucleus provides a separate compartment for transcription. Original RNA transcript (pre-mRNA) processed in various ways before leaving nucleus as mRNA

9. RNA RNA is used as an intermediary between DNA and proteins
RNA is a single strand nucleotide polymer
Composition
Sugar- Ribose
Phosphate group(s)
Uracil substitutes for thymine

10. uracil

11. Transcription Overview
A copy of the DNA is made in the form of mRNA (messenger RNA) in transcription

13. Translation Overview
Translation involves mRNA, tRNA (transfer RNA), and rRNA (ribosomal RNA) coordinating to produce proteins

14. Translation Overview 2
mRNA has sequences of 3 nucleotides called codons
Codons are read in sequences of 3 called triplet code

16. Translation Overview 3 Codons are written 5’ to 3’ fashion
Each codon codes for one amino acid
Codons do not overlap

17. Translation Overview 4
Four bases can combine in 43 combinations– more than enough to code for the 20 naturally occurring amino acids
43 = 64
Why don’t we have 64 amino acids?

18. Universal Code Genetic code (AA code) nearly universal
From simplest bacteria to complex animals
Genes can be transcribed & translated after being transplanted from one species to another
Tobacco plant with firefly gene

19. Translation Overview - tRNA
tRNA molecule has a sequence of 3 nucleotides- the anticodon
Anticodons base pair with the codon in a complementary way
Anticodons are written in 3’ to 5’ direction

21. Translation Overview 6 The E, P, and A are rRNA
Ribosomes are composed of proteins and rRNA
The E, P, and A are rRNA
Ribosome

22. Transcription: Initiation
Synthesis of RNA from the DNA template
Main enzyme is RNA polymerase
Transcription does not involve a primer - it begins at a promoter site
The promoter is a “start” sequence

24. Transcription: Elongation
RNA synthesis proceeds in a 5’-3’ direction copying DNA from the 3’-5’
Upstream- towards 5’ end of mRNA sequence
Downstream- towards the 3’ end of mRNA sequence

25. Transcription: Elongation
RNA polymerase moves along the DNA
Untwists the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

27. Transcription: Elongation
Bacterial promoters are about 40 bases long and are located in the DNA just upstream from the starting point

28. Transcription: Termination
Sequences at the end of the gene act as stop signals
Typically only one strand of DNA is transcribed and is called the template strand

30. Termination (eukaryotes)
Enzymes in nucleus modify pre-mRNA (before genetic messages sent to the cytoplasm)
mRNA contains additional base sequences that do not directly code for proteins
5’ end: modified nucleotide cap
3’ end: poly-A tail

31. `

32. 5’ Cap & Poly A Tail 5’ Cap Poly A Tail
Protect mRNA from hydrolytic enzymes
Functions as an “attach here” signal for ribosomes
Poly A Tail
50 – 250 nucleotides
Same function as 5’ cap
Faciliates export from nucleus

33. RNA Modification RNA splicing Removes introns and joins exons
Introns – noncoding regions of DNA
Exons – coding portions of DNA
Introns stay “in” the nucleus, exons “exit” the nucleus

35. Spliceosome Splicing accomplished by a spliceosome
Consists of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs)
Each snRNP has several protein molecules and a small nuclear RNA molecule (snRNA)
Each is about 150 nucleotides long

37. Translation 1 During translation, the nucleic acid message is decoded
An amino acid is attached to tRNA before becoming incorporated into a polypeptide
To form a polypeptide chain, the amino and carboxyl groups of amino acids are joined

40. Translation 2
The specific sequence of the amino acids (primary structure) is dictated by the sequence of codons of the mRNA

41. Translation 3
tRNA is linked to amino acids by aminoacyl-tRNA synthetases
This is an energy requiring process

42. RNA molecules - tRNA
RNA molecules have specialized regions with specific functions
tRNA molecules have attachment sites for amino acids

43. RNA molecules
tRNA molecules have anticodons that bind to complementary codons of the mRNA
If the mRNA codon is UAC, then what is the anticodon present on the tRNA?

44. RNA molecules
tRNA must be recognized by both the specific aminoacyl-tRNA synthetase and the ribosome

45. RNA molecules
tRNA ~ 70 nucleotides long, some generic sections & some unique sections
The nucleotide chain is folded back upon itself to form 3 or more loops with unpaired nucleotides exposed

46. Ribosomes
Components of translational machinery come together at the ribosomes
Ribosomes are composed of two subunits

47. Ribosomes
The large subunit has a groove into which the small subunit fits
Ribosomes are transcribed from DNA, but do not carry information
Function as physical site of translation & as a catalyst

48. Ribosome The A site of the ribosome is where the aminoacyl-tRNA binds
The P site is where the tRNA holding the polypeptide chain is positioned

49. Translation Steps
Inititiation
Elongation
Termination

50. Initiation
Initiation factors (proteins) move an initiation tRNA onto the small ribosomal subunit
The codon for the initiation is AUG, which codes for the amino acid methionine

51. Initiation 2
Initiation complex binds to ribosome recognition sequences on the mRNA, and aligns anticodon of tRNA with the codon of mRNA

52. Initiation 3
The large ribosomal subunit then binds, forming the functional ribosome

53. Elongation 1 Addition of new amino acids
Initiator tRNA is bound to P site of the ribosome - A site is unoccupied until the next aminoacyl-tRNA moves in

54. Elongation 2 Energy for this process comes from GTP
Peptide bond formation
Amino group of the new amino acid & carboxyl group of “old” amino acid

55. Elongation 3
Protein synthesis proceeds from the amino end to the carboxyl end
The tRNA molecule is released from the P site requiring ribozyme, peptidyl transferase
Translocation - movement of the growing polypeptide chain from the A site to P site - energy comes from GTP

56. Elongation 4
Translation of the mRNA proceeds in a 3’ to a 5’ direction, which is the same as the direction of transcription
Translocation ensures

57. Termination Occurs when the mRNA presents the codons UAA, UGA, or UAG;
No complementary tRNA
Release factors recognize codons
The ribosome dissociates into the two subunits

58. Translation Tutorial

59. Polyribosome
A single mRNA can make many copies of a polypeptide simultaneously
Multiple ribosomes, polyribosomes, may trail along the same mRNA

60. Mutations are changes in DNA
Point mutations – single bp change
Inheritable (if occurs in gametes)
Mutations are changes in DNA

61. Point mutation Base pair substitution Some have little/no impact
Replacement of a pair of complementary nucleotides with another nucleotide pair
Some have little/no impact
Silent mutation
AA change
Similar
Nonessential

62. Missense mutation
AA change

63. Nonsense mutation
AA change to stop codon

64. Mutations are changes in DNA
Frameshift mutations- involve the insertion or deletion of a base
They result in an entirely different sequence of amino acids because they change the _______________
Mutations are changes in DNA

65. Frameshift Mutations

66. Mutations are changes in DNA
Transposons - movable sequences of DNA that may move into another area of DNA
May disrupt genes, but may inactivate others
Transposons have some similarities to retroviruses
Mutations are changes in DNA

67. Transposons

68. Barbara McClintock (1902-1992)
Nobel Prize 1983
Cornell University
Discovered the transposons “jumping genes”
Studied maize

69. Mutations
Hot spots - regions of DNA more likely to undergo mutations, and are often regions of repeated nucleotides, causing the polymerases to slip

70. Mutagens Agents that cause mutations Including ionizing radiation
Mutations in somatic cells are not passed on to the next generation
Some mutagens are also carcinogens