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Molecular Biology of the Gene, 5/E --- Watson et al. (2004)

Part I: Chemistry and Genetics

Part II: Maintenance of the Genome

Part III: Expression of the Genome

Part IV: Regulation

Part V: Methods

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Part III: Expression of the Genome

This part concerned with one of the greatest challenges in understanding the gene －how the gene is expressed.

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Ch 12: Mechanisms of transcription

Ch 13: RNA splicingCh 14: TranslationCh 15: The genetic code

The revised central dogma

RNA processing

基因组的保持

基因组的表达

学时分配和教学日历内容内容 学时学时

课程简介－从相互认识到中心法则 6

DNA 和 RNA 的结构 3

基因组的保持： DNA 复制 6

基因组的表达：转录 （小考 1 ） 6

基因组的表达： RNA 剪接与转录后加工 6

基因组的表达：翻译与遗传密码（文献报告） 6

基因调控：原核调控（小考 2 ） 6

基因调控：真核调控 1 3

基因调控：真核调控 2 － RNA 干扰与 miRNA 调控（秀）

3

分子生物学技术 6

平时小考两次 3

CHAPTER 14CHAPTER 14

TranslationTranslation

•Molecular Biology Course

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What is translation?

--it is the story about decoding the genetic information contained in messenger RNA (mRNA) into proteins.

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Questions addressed in this chapter

What are the main challenges of translation and how do organisms overcome them?

What is the organization of nucleotide sequence information in mRNA?

What is the structure of tRNAs, and how do aminoacyl tRNA synthetases recognize and attach the correct amino acids to each tRNA?

How does the ribosome orchestrate the translation process?

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Translation extremely costs

In rapid growing bacterial cells, protein synthesis consumes

80% of the cell’s energy 50% of the cell’s dry weight

Why?

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The main challenges of translation The genetic information in

mRNA cannot be recognized by amino acids.

The genetic code has to be recognized by an adaptor molecule (translator), and this adaptor has to accurately recruit the corresponding amino acid.

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Translation machinery

1. mRNAs (~5% of total cellular RNA)

2. tRNAs (~15%)3. aminoacyl-tRNA synthetases ( 氨酰 tRNA合成酶 )

4. ribosomes (~100 proteins and 3-4 rRNAs--~80%)

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Outline Topics 1-4: Four components of

translation machinery. T1-mRNA; T2-tRNA; T3-Attachment of amino acids to tRNA (aminoacyl-tRNA synthetases); T4-The ribosome

Topic 5-6: Translation process. T5-initiation; T6-elongation; T7-termination.

Topic 8: Translation-dependent regulation of mRNA and protein stability

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Topic 1: mRNA

Only a portion of each mRNA can be translated.

The protein-coding region of the mRNA consists of an ordered series of 3-nt-long units called codons that specify the order of amino acids.

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1-1 polypeptide chains are specified by ORF

The protein coding region of each mRNA is composed of a contiguous, non-overlapping string of codons called an opening reading frame (ORF) .

Each ORF begins with a start codon and ends with a stop codon.

Messa

ge R

NA

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The start codon —the first codon of an ORFIn bacteria : AUG, GUG, or UUG (5’-3’)In eukaryotic cells: 5’-AUG-3’

Functions:1.Specifies the first amino acid to be

incorporated into the growing polypeptide chain.

2.Defines the reading frame for all subsequent codons.

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Prokaryotic mRNA (polycistrionic)

Eukaryotic mRNA (monocistrionic)

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Fig 14-1 Three possible reading frames of the E. coli trp leader sequence

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1-2 Prokaryotic mRNAs have a ribosome binding site that recruits the translational machinery

Messa

ge R

NA 1-3 Eukaryotic mRNA

are modified at their 5’ and 3’ ends to facilitate translation.

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Ribosome binding site (RBS) or SD-sequence in prokaryotic mRNA, complementary with the sequence at the 3’ end of 16S rRNA.

Fig 14-2-a structure of prokaryotic mRNA

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Once Kozak sequence

Eukaryotic mRNA uses a methylated cap to recruit the ribosome. Once bound, the ribosome scans the mRNA in a 5’-3’ direction to find the AUG start codon.

Kozak sequence increases the translation efficiency.

Poly-A in the 3’ end promotes the efficient recycling of ribosomes.

Fig 14-2-b

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Fig 1-29

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Topic 2: tRNA

At the heart of protein synthesis is the translation of nucleotide sequence information into amino acids. This work is accomplished by tRNA.

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1. The are many types of tRNA molecules in cell (~40).

2. Each tRNA molecule is attached to a specific amino acids (20) and each recognizes a particular codon, or codons (61), in the mRNA.

3. All tRNAs end with the sequence 5’-CCA-3’ at the 3’ end, where the aminoacyl tRNA synthetase adds the amino acid.

2-1: tRNA are adaptors between codons and amino acids

TR

AN

SFE

R R

NA

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1. tRNAs are 75-95 nt in length. 2. There are 15 invariant and 8 semi-invariant

residues. The position of invariant and semi-variant nucleosides play a role in either the secondary and tertiary structure.

3.3. There are many modified bases, which There are many modified bases, which sometimes accounting for 20% of the total sometimes accounting for 20% of the total bases in one tRNA molecule. Over 50 bases in one tRNA molecule. Over 50 different types of them have been different types of them have been observed.observed.

1. tRNAs are 75-95 nt in length. 2. There are 15 invariant and 8 semi-invariant

residues. The position of invariant and semi-variant nucleosides play a role in either the secondary and tertiary structure.

3.3. There are many modified bases, which There are many modified bases, which sometimes accounting for 20% of the total sometimes accounting for 20% of the total bases in one tRNA molecule. Over 50 bases in one tRNA molecule. Over 50 different types of them have been different types of them have been observed.observed.

Primary structure

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Fig 14-3 unusual bases

4. Pseudouridine (U) is a modified base. These modified bases in tRNA lead to improved tRNA function

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The cloverleaf structure is a common secondary structural representation of tRNA molecules which shows the base paring of various regions to form four stems (arms) and three loops.

2-2: tRNAs share a common secondary structure that resembles a cloverleaf

TR

AN

SFE

R R

NA

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Fig 14-4 the secondary structure

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2-3: tRNAs have an L-shaped 3-D structure

Fig 14-5 the 3-D structure of tRNA

D loop U loop

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Formation of the 3-D structure :

9 hydrogen bonds (tertiary hydrogen bonds) mainly involving in the base paring between the invariant bases help the formation of tRNA tertiary structure.

Formation of the 3-D structure :

9 hydrogen bonds (tertiary hydrogen bonds) mainly involving in the base paring between the invariant bases help the formation of tRNA tertiary structure.

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The cloverleaf structure of a tRNA. The tRNA is drawn in the conventional cloverleaf structure, with the different components labeled. 15 invariant nucleotides (A, C, G, T, U, Y, where =pseudouridine) and 8 semi-invariant nucleotides (abbreviations: R, purine; Y, pyrimidine) are indicated. Optional nucleotides not present in all tRNAs are shown as smaller dots. The standard numbering system places position 1 at the 5’ end and

position 76 at the 3’ end; not all of the optional nucleotides are included. The invariant and semi-invariant nucleotides are at positions 8, 11, 14, 15, 18, 19, 21, 24, 32, 33, 37, 48, 53, 54, 55,

56, 57, 58, 60, 61, 74, 75 and 76. The nucleotides of the anticodon are at positions 34, 35 and 36.

http://www.ncbi.nlm.nih.gov/books/

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Base pairing between residues in the D-and T-arms fold the tRNA molecule into an L-shape, with the anticodon loop at one end and the amino acid acceptor site at the other (Fig. 14-5). The base pairing is strengthened by base stacking interactions.

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Topic 3: attachment of amino acids to tRNA

Amino acids should be attached to tRNA first before adding to polypeptide chain.

tRNA molecules to which an amino acid is attached are said to be charged, and tRNAs lacking an amino acid are said to uncharged.

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3-1 tRNAs are charged by attachment of an amino acid to the 3’ terminal A of the tRNA via a high energy acyl linkage

Energy: The energy released when the high-energy bond is broken helps drive the peptide bond formation during protein synthesis.

Enzyme: Aminoacyl tRNA synthetase Aminoacyl tRNA synthetase catalyzing the reaction has three binding sites for ATP, amino acid and tRNA.

ATTA

CH

MEN

T O

F AM

INO

AC

IDS

TO

tRN

A

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3-2 Aminoacyl tRNA synthetases charge tRNA in two steps (reactions)(reactions)

1. Adenylylation (腺苷酰化 ) of amino acidsamino acids: transfer of AMP to the COO- end of the amino acids.

2. tRNAtRNA charging: transfer of the adenylylated amino acids to the 3’ end of tRNA, generating aminoacyl-tRNAs (charged tRNA).

ATTA

CH

MEN

T O

F AM

INO

AC

IDS

TO

tRN

A

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Class I: attach the amino acids to the 2’OH of the tRNA, and is usually monomeric.

Class II: attach the amino acids to the 3’OH of the tRNA, and is usually dimeric or tetrameric.

There are two classes of tRNA synthetases.

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Step 1-Adenylylation of amino acids: the aminoacyl-tRNA synthetase attaches AMP to the-COOH group of the amino acid utilizing ATP to create an aminoacyl (氨酰的 ) adenylate (腺苷酸 ) intermediate. As As a result, the adenylylated aa binds to the synthetase a result, the adenylylated aa binds to the synthetase tightlytightly. [This step is also called activation of amino acids p65]

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A class II tRNA synthetaseA class II tRNA synthetase

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Step 2- tRNA charging: transfer of the adenylated amino acid to the 3’ end of the appropriate tRNA via the 2’ or 3’-OH group, and the AMP is released as a result.

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Nature structural and Molecular Biology, 2005, 12:915-922

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3-3 ： each aminoacyl tRNA synthetase attaches a single amino acids to one or more cognate/appropriatecognate/appropriate tRNAs

Each of the 20 amino acids is attached to the appropriate tRNA (s) by aminoacyl-tRNA synthetases.

Most amino acids are specified by more than one codon, and by more than one tRNA as well.

ATTA

CH

MEN

T O

F AM

INO

AC

IDS

TO

tRN

A

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The same synthetase is responsible for charging all tRNAs for a particular amino acid (one synthetaseone amino acid).

Consequently, most organisms have 20 synthetases for 20 different amino acids.

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3-4 tRNA synthetases recognize unique structure features of cognate tRNAs

ATTA

CH

MEN

T O

F AM

INO

AC

IDS

TO

tRN

A

The recognition has to ensure two levels of accuracy: (1) each tRNA synthetase must recognizerecognize the correct set of tRNAs for a particular amino acids; (2) each synthetase must chargecharge all of these isoaccepting tRNAs isoaccepting tRNAs ( 即由一种synthetase所识别的不同 tRNAs)

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The specificity determinants for accurate recognition are clusters at two distinct sites: the acceptor stem and the anti-codon loop.

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Fig 14-8

Fig 14-7

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Identity elements (specificity determinants ) in various tRNA molecules

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3-5 Aminoacyl-tRNA formation is very accurate: selection of the correct amino acid

The aminoacyl tRNA synthetases discriminate different amino acids according to different natures of their side-chain groups.

ATTA

CH

MEN

T O

F AM

INO

AC

IDS

TO

tRN

A

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Fig. 14-9

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3-6 Some aminoacyl tRNA synthetase use an editing pocket to charge tRNAs with high accuracy.

ATTA

CH

MEN

T O

F AM

INO

AC

IDS

TO

tRN

A

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Isoleucyl tRNA synthetase as an example:1. Its editing pocket near the catalytic pocket allows it

to proof read the product of the adenylation reaction (step #1).

2. AMP-valine and other mis-bound aa can fit into this editing pocket and get hydrolyzed. But AMP-Ile is too big to fit in the pocket. Thus, the binding pocket serves as a molecular sieve to exclude AMP-valine etc.

Val

Ile

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Therefore, Ile-tRNA synthetase discriminates against valine twice: the initial binding and adenylylation of the amino acid, and then the editing of the adenylylated amino acid. Each step discriminates by a factor of ~100, and the overall selectivity is about 10,000-fold.

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1. Ribosome recognize tRNAs but not amino acids (how to prove?). 2. It is responsible to place the charged tRNAs onto mRNA through base pairing of the codon in mRNA and anticodon in tRNA.

3-7 Ribosomes is unable to discriminate between correctly or incorrectly charged tRNAs (是否携带正确的氨基酸 )

ATTA

CH

MEN

T O

F AM

INO

AC

IDS

TO

tRN

A

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Topic 4: the ribosome

1. Ribosome composition2. Ribosome cycle3. Peptide bond formation4. Ribosome structure

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4-1 the ribosome is composed of a large and a small subunit

The large subunit contains the peptidyl transferase center, which is responsible for the formation of peptide bonds.

The small subunit interacting with mRNA contains the decoding center, in which charged tRNAs read or “decode” the codon units of the mRNA.

RIB

OSO

MES

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Fig 14-13** Ribosome

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4-2: the large and the small subunits undergone association and dissociation during each cycle of translation.

RIB

OSO

MES

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Ribosome cycles: In cells, the small and large ribosome subunits associate with each other and the mRNA, translate it, and then dissociate after each round of translation. This sequence of association and dissociation is called the ribosome cycle.

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Fig 14-14 Overview of the events of translation/ribosome cycle

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Polysome/polyribosome: an mRNA bearing multiple ribosomes

• Each mRNA can be translated simultaneously by multiple ribosomes

Fig 14-15 A polyribosome

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RIB

OSO

MES

4-3 New amino acids are attached to the C-terminus of the growing polypeptide chain.

Protein is synthesized in a N- to C- terminal direction

4-4 Peptide bonds are formed by transfer of the growing peptide chain from peptidyl- tRNA to aminoacyl-tRNA.

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Fig 14-16

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The structure of the ribosome

4-5 Ribosomal RNAs are both structural and catalytic determinants of the ribosomes

4-6 The ribosome has three binding sites for tRNA.

4-7 Channels through the ribosome allow the mRNA and growing polypeptide to enter and/or exit the ribosome.

RIB

OSO

MES

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Fig 14-17 two views of the ribosome

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4-6 Three binding site for tRNAs

Fig 14-18

A site: to bind the aminoacylated-tRNAP-site: to bind the peptidyl-tRNAE-site: to bind the uncharged tRNA

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14-1

9 3

-D s

tru

cture

of

the

riboso

me incl

udin

g 3

boun

d t

RN

A

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4-7 Channel for mRNA entering and exiting are located in the small subunit (see Fig. 14-18)

There is a pronounced kink in the mRNA between the two codons at P and A sites. This kink places the vacant A site codon for aminoacyl-tRNA interaction.

Fig 14-20

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4-7 Channel for polypeptide chain exiting locates in the large subunit

The size of the channel only allow a very limited folding of the newly synthesized polypeptide

Fig 14-21

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Translation process

T5: Initiation of translationT6: Elongation of translationT7: termination of translation

Watch the animation on your study CD

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Questions

1. Compare the mechanism of translation initiation in prokaryotes and eukaryotes (similarity and difference)

2. How do aminoacyl-tRNA synthetases and the ribosomes contribute to the fidelity of translation, respectively?

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Overview of the events of translation

Termination Elongation

Initiation

Fig 14-14

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T5: Initiation of translation

Initiation in prokaryotic cells (1-3)

Initiation in eukaryotic cells (4-6)

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5-1 Prokaryotic mRNAs are initially recruited to the small subunit by base pairing to rRNA.

INIT

IATIO

N O

F TR

AN

SLA

TIO

N

Fig 14-23

RBS is also called Shine–Dalgarno sequence

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5-2 A specialized tRNA (initiator tRNA) charged with a modified methionine (f-Met) binds directly to the prokaryotic small subunit.IN

ITIA

TIO

N O

F TR

AN

SLA

TIO

N Fig 14-24

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5-3 Three initiator factors direct the assembly of an initiation complex that contains mRNA and the initiator tRNA.IN

ITIA

TIO

N O

F TR

AN

SLA

TIO

N

1. Formation of the 30S initiation complex: IFs1-3 + 30S + mRNA + fmet-tRNA.

2. Formation of the 70S initiation complex: 50S + 30S + mRNA + fmet-tRNA

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Eukaryotic initiation5-4 Eukaryotic ribosomes are recruited to the 5’ cap.

5-5 The start codon is found by scanning downstream from the 5’ end of the mRNA.

INIT

IATIO

N O

F TR

AN

SLA

TIO

N

Figs 14-26 and -27

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5-6 Translation initiation factors hold eukaryotic mRNAs in circles

Try to explain how the mRNA poly-A tail contributes to the translation efficiency?

INIT

IATIO

N O

F TR

AN

SLA

TIO

N

Fig 14-29

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T6: Translation elongation

1. Aminoacyl-tRNA binding to A site

2. Peptide bond formation

3. Translocation

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6-1 Aminoacyl-tRNAs are delivered to the A site by elongation factor EF-Tu

ELO

NG

ATIO

N O

F TR

AN

SLA

TIO

N

1.1. EF-Tu-GTP binds to EF-Tu-GTP binds to aminoacyl-tRNAsaminoacyl-tRNAs

2.2. Deliver a tRNA to A Deliver a tRNA to A site on ribosome site on ribosome

3.3. When correct codon-When correct codon-anticodon occurs, EF-anticodon occurs, EF-Tu interacts with the Tu interacts with the factor-binding center factor-binding center on ribosome and on ribosome and hydrolyzes its bound hydrolyzes its bound GTPGTP

4.4. EF-Tu-GDP leaves EF-Tu-GDP leaves ribosomeribosome

Fig 14-30

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6-2 The ribosome uses multiple mechanisms to select against incorrect aminoacyl-tRNAs

ELO

NG

ATIO

N O

F TR

AN

SLA

TIO

N

Additional hydrogen Additional hydrogen bonds are formed bonds are formed between two adenine between two adenine residues of the 16S residues of the 16S rRNA and the minor rRNA and the minor groove of the groove of the anticodon-codon pair anticodon-codon pair only when they are only when they are correctly paired. correctly paired.

Fig 14-31a

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Correct base pairing Correct base pairing allows EF-Tu interact allows EF-Tu interact with the factor with the factor binding center on binding center on ribosome, which is ribosome, which is important for GTP important for GTP hydrolysis and EF-Tu hydrolysis and EF-Tu release. release.

Fig 14-31b

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Only correct base paired Only correct base paired aminoacyl-tRNAs aminoacyl-tRNAs remain associated remain associated with the ribosome as with the ribosome as they rotate into the they rotate into the correct position for correct position for peptide bond peptide bond formation.formation.

Fig 14-31c

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6-3 Ribosome is a ribozyme ( 重点，催化如何发生？ )

ELO

NG

ATIO

N O

F TR

AN

SLA

TIO

N

Fig 14-32 Fig 14-16

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ELO

NG

ATIO

N O

F TR

AN

SLA

TIO

N

1.1.The role of L27 protein? The role of L27 protein? 2.2.The role of A2451 The role of A2451

nucleotide residue in the nucleotide residue in the 23 rRNA?23 rRNA?

3.3.The role of the 2’-OH of The role of the 2’-OH of the A residue at the 3’ of the A residue at the 3’ of the peptidyl-tRNA (a part the peptidyl-tRNA (a part of a proton shuttle)? of a proton shuttle)? Figure-14-33Figure-14-33

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6-4 & 5 Elongation factor EF-G drive translocation of the tRNAs and the mRNA by displacing the tRNA bound to the A site

ELO

NG

ATIO

N O

F TR

AN

SLA

TIO

N

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EF-G mimics a tRNA molecule so as to displace the tRNA bound to the A site

EF-G-GDPEF-Tu-GDPNP-Phe-tRNA

Fig 14-35

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6-6 EF-Tu-GDP and EF-G-GDP must exchange GDP for GTP prior to participating in a new round of elongation.

ELO

NG

ATIO

N O

F TR

AN

SLA

TIO

N

1.1. EF-G-GDP: GDP has a lower EF-G-GDP: GDP has a lower affinity, and GDP is released affinity, and GDP is released after GTP hydrolysis. The free after GTP hydrolysis. The free EF-G rapidly binds a new GTP.EF-G rapidly binds a new GTP.

2.2. EF-Tu-GDP requires a GTP EF-Tu-GDP requires a GTP exchange factor EF-Ts to exchange factor EF-Ts to displace GDP and recruit GTP displace GDP and recruit GTP to EF-Tu. (Fig. 14-36) to EF-Tu. (Fig. 14-36)

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Topic 7: Translation termination

1.1.Releasing factors act to Releasing factors act to release the synthesized release the synthesized peptide from the peptidyl peptide from the peptidyl tRNA in the ribosome.tRNA in the ribosome.

2.2.Ribosome recycling factor, Ribosome recycling factor, EF-Tu and IF3 act to EF-Tu and IF3 act to recycle the ribosome.recycle the ribosome.

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7-1 & 2 & 3 The action of Class I and II releasing factors

7-1 Class I and Class II releasing factors terminate translation in response to stop codons.

TER

MIN

ATIO

N O

F TR

AN

SLA

TIO

N

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7-2 Class I releasing factors (bacterial RF1 and RF2, eukaryotic eRF1) recognize stop codons by its peptide anticodon and trigger the release of the peptidyl chain by the conserved GGQ motif.(Figure 14-37). RF1 has a structure resembles tRNA (Figure 14-38), explaining why it can enters A site.

TER

MIN

ATIO

N O

F TR

AN

SLA

TIO

N

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7-3 Class II releasing factor remove Class I releasing factor from the A site. And this function is controlled by GDP/GTP exchange and GTP hydrolysis (Figure 14-39).

TER

MIN

ATIO

N O

F TR

AN

SLA

TIO

N

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7-4 Recycling of the ribosome by a combined effort of RRF (ribosome recycling factor), EF-Tu-GTP and IF3. (Figure 14-40)

TER

MIN

ATIO

N O

F TR

AN

SLA

TIO

N

RRF mimics a tRNA and enters A RRF mimics a tRNA and enters A site, and EF-Tu-GTP pushes it site, and EF-Tu-GTP pushes it into P site. This action pushes into P site. This action pushes the uncharged tRNAs from P-site the uncharged tRNAs from P-site and E-site. Then mRNA is and E-site. Then mRNA is dissociated from ribosome (on dissociated from ribosome (on direct interaction), and the small direct interaction), and the small subunit is sequestered by IF3.subunit is sequestered by IF3.

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Topic 8: translation dependent regulation of mRNA and

protein stability

Here regulation refers cellular processes that deal with defective mRNA and their translated product.

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8-1: The SsrA RNA rescues ( 拯救 ) ribosomes that translate broken mRNAs lacking a stop codon (prokaryotes)

1.The ribosomes are trapped or stalled on the broken mRNA lacking a stop codon

2.The stalled ribosomes are rescued by the action of a chimeric RNA molecule that is part tRNA and part mRNA, called tmRNA.

3.SsrA is a 457-nt tmRNA

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Fig 14-39 SsrA rescues the stalled ribosomes

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8-2: Eukaryotic cells degrade mRNAs that are incomplete or have premature stop codons.

Translation is tightly linked to the process of mRNA decay in eukaryotic cells

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When an mRNA contains a premature stop codon (nonsense codon), the mRNA is rapidly degraded by nonsense mediated mRNA decay.

Pre-releasing the ribosome at the nonsense codon prior to reaching the exon-junction complex initiates a talk between the complex and ribosome to remove the 5’ cap from the mRNA

Nonsense mediated mRNA decay

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1. Translation of a normal eukaryotic mRNA displace all the exon junction complex

Fig 14-40a

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2. Nonsense mediated mRNA decay

Fig 14-40b

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Non-stop mediated mRNA decay rescues ribosomes that translate mRNAs lacking a stop codon.

(1)The lack of a stop codon results in ribosome translation into the poly-A tail to produce poly-Lys at the C-terminus of the polypeptide; the poly-Lys marks the newly synthesized for rapid degradation.

Nonstop mediated decay

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(2)The ribosome eventually stalls at the 3’ end of the mRNA, which is bound by the Ski7 protein that triggers the ribosome dissociation and recruits a 3’-5’ exonuclease activity to degrade the “nonstop” mRNA.

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1. The main challenge of translation

2. The structure and function of four components of the translation machinery. ( 重点 )

3. Translation initiation, elongation and termination ( 具体过程和翻译因子的作用 - 注意起始阶段原核与真核的不同，重点 )

4. Translation-dependent regulation of the stability of defective mRNAs and the resulted protein ( 生物学问题是什么，在原核和真核分别怎么解决的 )

Key points of the chapter