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 sitesnRNP 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

28S

5.8S5S

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]