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BIOL 200 (Section 921) Lecture # 4-5, June 22/23, 2006. UNIT 4: BIOLOGICAL INFORMATION FLOW - from DNA (gene) to RNA to protein Reading : - PowerPoint PPT Presentation
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BIOL 200 (Section 921)Lecture # 4-5, June 22/23, 2006
• UNIT 4: BIOLOGICAL INFORMATION FLOW - from DNA (gene) to RNA to protein
• Reading: ECB (2nd Ed.) Chap 7, pp. 229-262; Chap 8, pp. 267-286 (focus only on parts covered in lecture). Related questions 7-8, 7-9, 7-11, 7-12, 7-14, 7-16, 7-17, 8-5a,b,d; orECB (1st ed.) Chap 7, pp. 211-240; 263-4; Chap 8, pp. 257-270 (focus only on parts covered in lecture). Related questions 7-9, 7-10, 7-12, 7-13, 7-15, 7-18, 7-19; 8-7b,c,e.
Lecture # 4,5: Learning objectives
• Understand the structural differences between DNA and RNA, and the directionality of transcription.
• Understand the structure of a eukaryotic gene. • Describe the general mechanism of transcription, including
binding, initiation, elongation and termination. Discuss factors regulating transcription.
• Describe processing for all three types of RNA, and discuss why it must occur.
• Describe the structural features of ribosomes.• Describe the genetic code and how it is related to the
mechanisms of transcription and translation. • Describe the coupling of tRNA and discuss the specificity of
amino acid tRNA synthetases.• Describe the general mechanism of translation including
binding, initiation, elongation and termination.
Transcription-same language (nuc. to nuc.)
Translation-different languages (nuc. to protein)
Genetic information flow [Fig. 7-1]
Eucaryotic vs. Procaryotic Biol. Info Flow [Fig. 7-20]
DNA vs. RNA: Differences in chemistry [Fig. 7-3]
DNA vs RNA (Lehninger et al. Biochemistry)
DNA vs. RNA: Intramolecular base pairs in RNA [Fig. 7.5] and Intermolecular base pairs in DNA [Fig.
5-7] assist their folding into a 3D structure
RNA DNA
Fig. 7-12, p. 219Procaryotic vs. eucaryotic transcription [Fig. 7-12]
In procaryotes, clusters of genes called operons can be carried by one mRNA [Fig. 8-6]
Fig. 7-9:Parts of a gene [Fig. 7-9]
Promoter sequence
Transcription start site
terminator
DNA template
Transcription factor binding to DNA [Fig. 8-4]
What types of bonds can you identify between this DNA and protein?
Eukaryotic transcription factors promote RNA Polymerase II binding [Equiv. Fig. 8-10, 2nd Ed.]
Directions of transcription along a short portion of bacterial chromosome [Fig. 7-10, p. 218]
3’
3’
5’
5’
DNA always read 3’ to 5’!
RNA always made 5’ to 3’!
RNA Polymerase: (1) Unwinds and rewinds DNA;(2) Moves along the DNA one nucleotide at a time; (3) Catalyzes The formation of phosphodiester bonds from the energy in the Phosphate bonds of ATP, CTR, GTP and UTP.
Transcription by RNA polymerase in E. coli [Lehninger et al. Principles of Biochemistry]
Fig. 7-37
introns exons
preRNA
5’ cap
polyA tailsplicing
mature mRNA
DNA in nucleus
Fig. 7-12: mRNAs in eukaryotes are processed
Prokaryote
mRNA
5’ cap3’ tail
Eukaryote
mRNA
Fig 7-12B
5’ cap
Functions:- Increased stability- Facilitate transport- mRNA identity
Bacterial vs. Eukaryotic genes [Fig. 7-13]
RNA splicing removes introns [Fig. 7-15]
RNA splicing: (1) done by small nuclear ribonuclear protein molecules [snRNPs, called as SNURPs] and other proteins [together called SPLICEOSOME], (2) Specific nucleotide sequences are recognized at each end of the intron
Fig. 7-17
RNA Splicing by SnRNP+proteins=spliceosome
exon1
intron
5’splice site snRNP 3’ splice site
lariat formation
Fig. 7-17
RNA Splicing
Spliceosome
Lariat=loop of intron
Exons joined
Mature mRNAexons joined together-introns removed
Removal of intron in the form of a lariat [Fig. 7-16]
Detailed mechanism of RNA splicing [Fig. 7-17]
Is RNA splicing biologically important or a wasteful process?
• Pre-mRNA can be spliced in different ways (alternate RNA splicing), thereby generating several different mRNAs which code for several different proteins [e.g. approx. 30,000 human genes can produce mRNAs coding for more than 100,000 proteins]
• Introns quickens the evolution of new and biologically important proteins e.g. three exons of the β-globin gene code for different structural and functional regions (domains) of the polypeptide
Alternative splicing can produce different proteins from the same gene [Fig. 7-18]
Becker et al. The World of the Cell
Transcription of two genes seen be EM [Fig. 7-8]
Becker et al. The World of the Cell
Biological information flow in procaryotes and eukaryotes [Fig. 7-20]
Specialized RNA binding proteins (e.g. nuclear transport receptor) facilitate export of mature mRNA
from nucleus to the cytoplasm [Fig. 7-19]
rRNA processing [Fig. 6-42 in Big Alberts]
45S transcript
18S 5.8S 28S
5S made elsewhereSmall
ribosomal subunit
Large ribosomal subunit
Processing of transfer RNA (tRNA) [Becker et al. The World of the Cell]
From RNA to Protein: translation of the information
• Translation: conversion of language of 4 nucleotides in mRNA into language of 20 amino acids in polypeptide
• Genetic code: a set of rules (triplet code) which govern translation of the nucleotide sequence in mRNA into the amino acid sequence of a polypeptide
• Codon: a group of three consecutive nucleotides in RNA which specifies one amino acid (e.g. ‘UGG’ specifies Trp and ‘AUG’ specifies Met)
Genetic code - triplet, universal, redundant and arbitrary [Fig. 7-21]
mRNA codons
Corresponding amino acid that will be attached onto tRNA
mRNA can be translated in three possible reading frames
“START” and “STOP” codons on mRNA are essential for proper mRNA translation
[Becker et al. The World of the Cell, Fig. 22-6]
Genetic code problem:
5’ AGUCUAGGCACUGA 3’For the RNA sequence above, indicate the amino acids that are encoded by the three possible reading frames.
If you were told that this segment of RNA was close to the3’ end of an mRNA that encoded a large protein, would you know which reading frame was used?
Frame 1-AGU CUA GGC ACU GAFrame 2-A GUC UAG GCA CUG AFrame 3-AG UCU AGG CAC UGA
Frame 1-Ser – Leu – Gly - Thr
What are the main players involved in the process of translation
1. Ribosomes [“protein synthesis machines”]: carry out the process of polypeptide synthesis
2. tRNA [“adaptors”]: : align amino acids in the correct order along the mRNA template
3. Aminoacyl-tRNA synthetases: attach amino acids to their appropriate tRNA molecules
4. mRNA: encode the amino acid sequence for the polypeptide being synthesized
5. Protein factors: facilitate several steps in the translation
6. Amino acids: required as precursors of the polypeptide being synthesized
Fig. 7-28
Eukaryotes
Large=60S
Small=40S
Prokaryotes
Large=50S
Small=30S
Ribosome Assembly
Centrifugation-separates organelles and macromolecules (panel 5-4)
Fixed angle Swinging bucket
Heavy particles move to pellet, e.g. nucleus
Svedberg unit, S, shows how fast a particle sediments when subject to centrifugal force.
From Becker, World of the Cell, p. 327
Fig. 7-23: tRNA structureOld ‘cloverleaf’ model 3-D L-shaped
modelAmino acid attachment
anticodon
H-bonds between complementary bases
Aminoacyl tRNA synthase charge-links tRNA with amino acids [Fig 7-26]
HighEnergyEster bond
Fig. 7-26, part 2
tRNA anticodon
binds mRNA codon
Fig. 7-28
18S
small subunit
28S5.8S5S
large subunit
Ribosomes made of rRNA and protein
Come together during translation
Ribosomes are protein synthesis factories [Fig. 7-29]
Ribosomes can be free in the cytosol or attached to the ER membranes
Fig. 7-35: polyribosomes or polysomes
Fig. 7-32: Initiation of translation
Start codon
Large subunit binds
Initiator rRNA-Met
+small subunit
Fig. 7-32: translation
Aminoacyl-tRNA binds to A site
Initiator tRNA in P site
Peptide bond forms
Ribosome shift: one codon
tRNA1 moves from P site to E site; tRNA2 moves from A site to P site
Fig. 7-30:
Elongation of polypeptide
Bind
Bond
Shift
Bind: aminoacyl-tRNA anticodon to mRNA codon
Bond: make peptide bond
Shift: move ribosome along mRNA, move tRNAs
Fig. 7-34: termination Stop codon at A site
Fig. 7-34:termination
stop codon at A site
Release C terminal from tRNA+shift
Release ribosome from mRNA
Release factor binds
Protein destruction• Proteolysis: digestion of proteins by
hydrolyzing peptide bonds• Proteases: enzymes that digest proteins• Many proteases aggregate to form a
proteasome in eukaryotes• Proteins are tagged for destruction by a
molecule called ubiquitin• Several proteins are also degraded in
lysosomes in eukaryotes
Proteasome-mediated degradation of short-lived and unwanted proteins [Fig. 7-36]
Mechanism of ubiquitin-dependentprotein degradation [Becker et al. The World of the Cell, Fig. 23-38]
SUMMARY1. Transcription2. RNA processing3. Translation4. Protein destruction
1. Where are Polymerases?2. Where are transcription start/stop sites?3. Where are the 3’ and 5’ ends of the transcript?4. Which direction are the RNA polymerases moving?5. Why are the RNA transcripts so much shorter than the length
of the DNA that encodes them?
Transcription of two genes seen be EM [Fig. 7-8]