chapter 8gene expression central dogma transcription translation dnamrnaprotein taking genetic...

Chapter 8 Gene Expression

Central Dogma

transcription translation

DNA mRNA protein

taking genetic information and using it to produce phenotypic traits

templatestrand

modificationgene

product

phenotype

8.1 Polypeptide chains are linear polymers of amino acids.

proteins:

catalyzing reactions (enzymes)regulating gene expression (regulatory proteins)structural proteins

one or more chains of amino acids (20)linked by peptide bonds

polypeptide chains

amino acids

carboncarboxyl group -COOHamino group -NH2

side chain -R

connected to each other betweencarboxyl group and amino group

(dehydration synthesis)

Fig. 8.1. Amino acid structure

Fig. 8.2. Chemical structures of amino acids specific in the genetic code

Fig. 8.3. Properties of a polypeptide chain

protein folding

interactions between amino acidsfolding to give 3-D structure

domains

picture of beta chain of hemoglobinshowing folding/domains

protein folding

interactions between amino acidsfolding to give 3-D structure

some proteins are made of multiple chainseach one being a subunit

domains

picture of hemoglobin

domain observations

vertebrate genomes have few protein domainsnot found in other organisms…

…but they are more complex because they haveput them together in more complex ways

only 7% of human proteins/domainsare specific to vertebrates

complexity is ~1.8 x fly or worm~5.8 x yeast

8.2 linear order of amino acids is encoded in the DNA.

most genes code of a single polypeptide (protein)

order of nucleotides determines order of amino acids

genes and proteins are colinear

8.2 linear order of amino acids is encoded in the DNA.

Chapter 8 Gene Expression REVIEW

8.3 DNA sequence determines RNA sequence.

synthesis of RNA is similar to that of DNA

•RNA is made from single stranded DNA•monomers are ribonucleotides A, C, G and U

Fig. 8.5. Structural differences between ribose and deoxyribose and between uracil and thymine

synthesis of RNA is similar to that of DNA

•RNA is made from single stranded DNA•monomers are ribonucleotides A, C, G and U•sequence of bases is determined by DNA sequence•nucleotides connected 5’-P to 3’-OH•nucleotides only added at the 3’ end of RNA•enzyme is different - RNA polymerase(s)

can initiate without a primer

Fig. 8.6A, B. RNA synthesis

RNA polymerases

prokaryotes - RNA polymerase holoenzyme six polypeptide chains

can process more than 104 nucleotides(while associated with the template)

processivity

RNA polymerases

eukaryotes - larger, more subunits

RNA polymerase IRNA polymerase IIRNA polymerase III

makes rRNAmRNA, snRNA’s, processingtRNA’s, 5S rRNA

processivity > 106 nucleotides

Transcription

which strandwhere to startwhere to stop ?

•promoter recognition•chain initiation•chain elongation•chain termination

which strandwhere to start

RNA polymerase binds to promoter

regions of DNA, 20-200 bp “recognized” by RNA polymeraseconsensus sequences (see fig. 8.8)

•promoter recognitionTranscription

Fig. 8.8. Base sequences in promoter regions of several genes in E. coli

binding strength varies~ closer to consensus has stronger binding

(Eukaryotes also have enhancers that interact with promoters)

*TATA box

•promoter recognition•chain initiation

Transcription

after RNA polymerase bindingtranscription begins at +1

only one strand is transcribed

•promoter recognition•chain initiation•chain elongation

Transcription

next nucleotide added to 3’ endRNA made in 5’ to 3’ direction

about 17 bp of DNA are separateddouble helix reformsRNA trails off as separate strand

Fig. 8.6C. RNA synthesis

Transcription

special DNA sequencesRNA polymerase dissociates from DNA

self termination sequence only

Fig. 8.9. (A) Base sequence of a transcription termination region; (B) the 3' terminus of an RNA transcript

which strand ?

RNA polymerase terminates transcription when loop forms in transcript

RNA sequence ?

Transcription

Fig. 8.10. EM of part of newt DNA showing tandem repeats of genes . [Courtesy of Oscar Miller and Barbara R. Beatty]

Transcription

special DNA sequencesRNA polymerase dissociates from DNA

self termination sequence only

termination protein sequence and protein

Transcription

mutations

in coding regionin promotorin termination sequence

change amino acidsno transcript ?long transcript ?

Transcription

only one strand is transcribedmight be either one(either strand can have promoters/terminators)

genes usually don’t overlap

Fig. 8.11. Typical arrangement of promoters and termination sites in a segment of a DNA molecule

5’ 3’

3’ untrans-lated region

5’ untrans-lated region

open reading frame(ORF)

RNA transcript is called the primary( 1°) transcript

in prokaryotes:used as mRNA directly for protein synthesisshort lifetime (minutes)

in eukaryotes:primary transcript is processed to become mRNAlonger lifetime (hours to days)

8.4 Eukaryotic 1° transcript is processed to become mRNA

RNA processing

1. terminal cap is added

at 5’ endadd modified guanosine5’ to 5’ linkageneeded for mRNA to bind to ribosome

RNA processing

1. terminal cap is added2. poly-A tail is added

add up to 200 A to the 3’ end

RNA processing

1. terminal cap is added2. poly-A tail is added3. remove introns

take out unnecessary RNAresplice needed RNA

5’ 3’

exon exon exonintron intron

Fig. 8.12. mRNA processing in eukaryotes

RNA processing

Many steps involved in processing are coupled

For example:

proteins involved with RNA polymerase to promote elongation also help recruit splicing machinery

the splicing machinery helps to:speed up elongationrecruit the polyadenylation machinery

RNA splicing (in the nucleus)

takes place at spliceosomesnuclear particlesprotein and small RNA’sforming snRNP’s

smallnuclearribo-nucleo-proteinparticles

U1, U2, U4, U5, U6

5 snRNP RNA:

U1, U2, U4, U5, U6

U1 binds to both ends of the intron and brings them together

U4 and U6 are normally paired, U2 is stable alone

U2 also binds to 3’ end of intron

U2 destabilizes U4-U6 complexand displaces U4 (U2 binds to U6)

U1, U2, U4, U5, U6

U4 and U6 are normally paired, U2 is stable alone

Fig. 8.13 . Interactions between small nuclear RNAs in snRNPs that are involved in splicing

Fig. 8.14B. Drawing of DNA-RNA hybrid

RNA splicing

hybridize DNA with processed RNA(denature / renature)

RNA splicing (in other places)

mitochondria happens w/out spliceosomesTetrahymena self slicing RNA

ribozymes

Table 8.2. Characteristics of human genes

titin has 178

typical is about 87 bp

BRAC1 has 21 intronsspread over 100,000 bmRNA = 7800 bpeptide has 1863 a.a.

RNA splicing

human genes are spread outhave small exons separated

by long introns

only about 5% of a gene codes for protein

longest human gene is muscle protein, dystrophin2.4 Mb (79 exons)codes for over 3500 amino acids

RNA splicing

many exons correspond to domains of the assembled protein

suggests that some current genes may have been assembled from smaller pieces

many genes more proteins?

a single primary transcript can be spliced in different ways to give different mRNA (thus different proteins)

sxl-protein+

http://fig.cox.miami.edu/~cmallery/150/gene/split_genes.htm

non-functionalprotein

http://departments.oxy.edu/biology/Stillman/bi221/111300/processing_of_hnrnas.htm

tropomyosin

8.5 Translation takes place on a ribosome

protein production includes two processes:

information transfergetting the amino acids in the correct order

chemical synthesishooking the amino acids together

protein production has 5 major components

•mRNA -

•ribosomes -

•tRNA -

•aminoacyl-tRNA synthetases -

•factors -

needed for assembly of ribosomehas information for amino acid sequence

2 subunits, align tRNA’s, attach a.a.’s

carry appropriate amino acid, have anticodon

puts a.a.’s on tRNA

for initiation, elongation and termination

Overview:

mRNA binds to ribosometRNA’s are brought in one by one with a.a.adjacent amino acids are joinedfinished protein is released from ribosome

initiationelongation

termination

Eukaryotic initiation:

eIF = eukaryotic Initiation Factorsnot elongation factors (pg. 294)

eIF4F binds to 5’ cap of mRNArecruits eIF4A and eIF4B

Fig. 8.15. Initiation of protein synthesis

creates binding site for:

eIF2, eIF3, eIF5, tRNAMet

small 40S subunit of ribosomemaking initiation complex 48S

creates binding site for:

eIF2, eIF3, eIF5, tRNAMet

small 40S subunit of ribosomemaking initiation complex 48S

scans for AUGeiF5 causes release of initiation factors

and recruitment of the 60S subunit

Ribosome (60S subunit) has three binding sites

Peptidyl

Aminoacyl

Fig. 8.15. Initiation of protein synthesis

hydrogen bonding between codon and anticodon

Elongation (three steps)

•bring in next tRNA (with amino acid)

•form new peptide bond

•move to next codon on mRNA

Energy for elongation is provided by: EF-2EF-1

- GTP- GTP

Elongation

1. 40S subunit shifts one codon “down” the messagenew “charged” tRNA is brought to A site

2. coupled reaction forms new peptide bond(peptidyl transferase activity)

3. large subunit moves to “catch up” to small subunittRNA’s are shifted

1. from P and E site1. to the A and P site

Elongation

completed one cyclerepeat for next codon

Elongation

eukaryotes

40S60S12-15 aa/sec

EF-1EF-2

prokaryotes

30S50S20 aa/sec

EF-TuEF-G

Terrmination (release phase)

eukaryotic termination codons:

UAGUAAUGA

prokaryotes

UAAUAG

UAAUGA

Fig. 8.18. Termination of protein synthesis

InitiationElongationTermination

protein folding

most proteins fold as they are being synthesized

aa with hydrophilic R surfaceaa with hydrophobic R internal

-helix-pleated sheet

alpha helix

http://wiz2.pharm.wayne.edu/biochem/nsphelix1.jpg

beta pleated sheet

http://www.sciencecollege.co.uk/SC/biochemicals/bsheet.gif

Fig. 8.19. A "ribbon" diagram of the path of the backbone of a polypeptide. [Adapted from W. I. Weiss, et al. 1992. Nature 360: 127.]

Fig. 8.20. Alternative pathways in protein folding

eukaryotic prokaryotic

one protein / mRNAreads from 5’cap - to termination codon

may be polycistronic(multiple proteins / mRNA)

can initiate in other areasAGGAGG

(Shine-Dalgarno sequence)

Fig. 8.21. Products translated from a three-cistron mRNA molecule

for example, 10 enzymes needed for histidine synthesis - one mRNA

Fig. 8.22. Direction of synthesis of RNA and of protein

by convention:write from L to RDNA 5’ to 3’protein amino to carboxyl

8.6 Genetic code for amino acids is a triplet code

list of all codons and amino acids they encode

4 =4x4 =

4x4x4 =

Fig. 8.23. Reading bases in an RNA molecule

codons are linear and non-overlapping

Fig. 8.24. Change in an amino acid sequence of a protein caused by the addition of an extra base

frameshift mutationreading frame

Fig. 8.25. Interpretation of the rll frameshift mutations

make synthetic polynucleotides

AAAAAAAAAAAA…UUUUUUUUUUUU…CCCCCCCCCCCC…GGGGGGGGGGGG…

translate in vitro and look at peptides made

Lys Lys Lys Lys…Phe Phe Phe Phe…Pro Pro Pro Pro…Gly Gly Gly Gly…

Fig. 8.26. Polypeptide synthesis in three different reading frames

redundancymore than one way to get most amino acids

universality (almost)minor differences in some protozoanssome organelles

tRNAs (how many different ones?)

small, single stranded RNA70-90 nucleotides long

5’ is monophosphate (instead of triphosphate)

folds on itself

anticodon region3’ end for attachment of a.a.

Fig. 8.27. tRNA cloverleaf configuration

Fig. 8.28B. Diagram of the three-dimensional structure of yeast tRNAPhe

“wobble” at the third position

number of distinct tRNAs is less than the # of codons

Table 8.4. Wobble rules for tRNAs of E. coli and S. cervisiae

8.7 Multiple ribosomes can move in tandem on mRNA

After ribosome has moves about 75 nucleotides another ribosome can initiate translation on the same message

in prokaryotes (no nucleus)

can have simultaneous transcription and translation

8.7 Multiple ribosomes can move in tandem on mRNA

http://www.phschool.com/science/biology_place/biocoach/images/translation/polysome.gif

chapter 8gene expression central dogma transcription translation dnamrnaprotein taking genetic...

chains of amino acids

linear polymers of amino

linear order of amino

rna synthesis slide

u slide

polypeptide chain slide

colinear slide

foldingdomains slide

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