Chapter 2 3 protein biosynthesis

04/25/2023 by TDLV 3Protein Synthesis • Genetic information: the form of specific sequences of nucleotides along the DNA strands • The DNA inherited leads to specific traits by dictati

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Protein biosynthesis

The Central Dogma of Life

replication

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Protein Synthesis

• Genetic information: the form of specific

sequences of nucleotides along the DNA strands

• The DNA inherited leads to specific traits by

dictating the synthesis of proteins

• Protein Synthesis: includes two stages:

transcription and translation

Transcription and Translation

• Transcription

– the synthesis of RNA under the direction of DNA

– Produces messenger RNA (mRNA)

• Translation

– the actual synthesis of a polypeptide under the direction

of mRNA– Occurs on ribosomes

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Transcription and Translation

• In prokaryotes, transcription & translation

occur together

Prokaryotic cell In a cell lacking a nucleus, mRNA

produced by transcription is immediately translated without additional processing.

(a)

TRANSLATION

TRANSCRIPTION DNA

mRNA Ribosome

Polypeptide

Transcription and Translation

• In a eukaryotic cell, the nuclear envelope separates

transcription from translation

• Extensive RNA processing occurs in the nucleus

Eukaryotic cell The nucleus provides a separate

compartment for transcription The original RNA transcript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA.

Polypeptide

Ribosome Nuclear envelope

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Transcription

• Product/catalysis/materials/

steps

• Transcription is the

DNA-directed synthesis of mRNA

• RNA synthesis

– catalyzed by RNA polymerase

– Follows the same base-pairing

rules as DNA, except that in

RNA, uracil substitutes for

thymine

TRANSCRIPTION - OVERVIEW

 Transcription

- when a cell requires a particular protein  specific mRNA synthesized

- first, a section of DNA containing the gene unwinds

- only one of the DNA strands copied (at the initiation point: sequence TATAAA)

- RNA polymerase: moves along DNA template in the 3’-5’direction  replicates DNA sequence into a complementary sequence of mRNA

- mRNA synthesized using complementary base pairing with uracil (U) replacing thymine (T)

 moves out of the nucleus to ribosomes in the cytoplasm, acts as the template for protein

biosynthesis (translation) and the DNA re-winds

 released at the termination point

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RNA

• Types/functions

• RNA is single stranded, not double stranded like DNA

• RNA is short, only 1 gene long, where DNA is very long and contains

many genes

• RNA uses the sugar ribose instead of deoxyribose in DNA

• RNA uses the base uracil (U) instead of thymine (T) in DNA.

Synthesis of an RNA Transcript

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Synthesis of an RNA Transcript - Initiation

• Promoters signal the

initiation of RNA synthesis

• Transcription factors help

eukaryotic RNA polymerase

recognize promoter

sequences

• A crucial promoter DNA

sequence is called a TATA

box

TRANSCRIPTION RNA PROCESSING TRANSLATION

DNA Pre-mRNA mRNA

Ribosome

Polypeptide

T A T A A AA ATAT T T T

DNA strand

5

Transcription factors

2

Additional transcription factors

3

Synthesis of an RNA Transcript - Elongation

Elongation

RNA polymerase

Non-template strand of DNA

3

5

5

Newly made RNA

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Synthesis of an RNA Transcript - Termination

• Specific sequences in the DNA signal

termination of transcription (TAA, TAG, TGA)

• When one of these is encountered by the

polymerase, the RNA transcript is released

from the DNA and the double helix can zip

up again.

Post Termination RNA Processing

• Most eukaryotic mRNAs aren’t ready to be translated into protein directly after being transcribed from DNA mRNA requires processing.

• RNA processing occur in the nucleus After this, the messenger RNA moves to the

cytoplasm for translation.

• The cell adds a protective cap to one end, and a tail of A’s to the other end

These both function to protect the RNA from enzymes that would degrade

• Most of the genome consists of non-coding regions called introns

– Non-coding regions may have specific chromosomal functions or have regulatory

purposes

– Introns also allow for alternative RNA splicing

• Thus, an RNA copy of a gene is converted into messenger RNA by doing 2 things:

– Add protective bases to the ends

– Cut out the introns

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Alteration of mRNA Ends

• Each end of a pre-mRNA molecule is modified in a particular way

– The 5 end receives a modified nucleotide cap

– The 3 end gets a poly-A tail

A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotidesadded to the 3 end

Protein-coding segment Polyadenylation signal

Poly-A tail

3 UTR Stop codon

TRANSLATION Ribosome

Polypeptide

G P P P

RNA Processing - Splicing

• The original transcript

from the DNA is called

pre-mRNA

• It contains transcripts of

both introns and exons

• The introns are removed

by a process called splicing

to produce messenger

RNA (mRNA)

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RNA Processing - Splicing

• Ribozymes are catalytic RNA molecules that function

as enzymes and can splice RNA

• RNA splicing removes introns and joins exons

TRANSCRIPTION

RNA PROCESSING

DNA Pre-mRNA mRNA

5 Cap

3 UTR 3 UTR

Pre-mRNA

mRNA

RNA Processing - by spliceosomes

• RNA Splicing can also be carried out by spliceosomes

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Alternative Splicing (of Exons)

• How is it possible that there are millions of human antibodies (proteins) when there are only about

30,000 genes?

• Alternative splicing refers to the different ways

the exons of a gene may be combined, producing different forms of proteins within the same gene- coding region

• Alternative pre-mRNA splicing is an important

mechanism for regulating gene expression in

higher eukaryotes

Protein – modular structure

• Proteins often have a modular architecture

consisting of discrete structural and functional

regions called domains

• In many cases, different exons code for the

different domains in a protein

Gene DNA

Exon 1 Intron Exon 2 Intron Exon 3 Transcription

RNA processing Translation

Domain 3

Domain 1 Domain 2

The Genetic Code

• Genetic information is encoded as a sequence of nonoverlapping base triplets, or codons

• The gene determines the sequence of bases along the length of an mRNA molecule

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The Genetic Code

• Codons: 3 bases code for the production of a specific

amino acid, sequence of three of the four different

nucleotides

 there are 4 x 4 x 4 = 64 possible codons

• 64 codons but only 20 amino acids  1 amino acid has

more than 1 codon to encode

• 3 of the 64 codons are used as STOP signals (TAA, TAG,

TGA); they are found at the end of every gene and mark

the end of the protein

• One codon is used as a START signal (ATG): it is at the start

of every protein

The Genetic Code

• A codon in messenger RNA is either translated into an amino acid or serves as a translational start/stop signal

Second mRNA base

Met or start

Phe Leu Leu

lle

Val

UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG

CAU CAC CAA CAG

CGU CGC CGA CGG AAU

AAC AAA AAG

AGU AGC AGA AGG GAU

GAC GAA GAG

GGU GGC GGA GGG

UGG

UAA UAG StopStop UGA StopTrp

His Gln Asn Lys Asp

Arg

Ser Arg Gly

U C A G U C A G U C A G U C A G

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Transfer RNA

• Consists of a single RNA strand that is only about 80 nucleotides long.

on the other end.

• A special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids.

• tRNA brings the amino acids to the ribosomes.

The “anticodon” is the 3 RNA bases that

matches the 3 bases of the codon on the

mRNA molecule

Transfer RNA

• 3 dimensional tRNA molecule is roughly “L” shaped

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Ribosomes

• Ribosomes facilitate the specific coupling of tRNA anticodons with

mRNA codons during protein synthesis

• The 2 ribosomal subunits are constructed of proteins and RNA

molecules named ribosomal RNA or rRNA

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Building a Polypeptide

Building a Molecule of tRNA

• A specific enzyme called an aminoacyl-tRNA synthetase

joins each amino acid to the correct tRNA

• Once the start codon has been identified, the ribosome

incorporates amino acids into a polypeptide chain

• RNA is decoded by tRNA (transfer RNA) molecules, which each transport specific amino acids to the growing chain

• Translation ends when a stop codon (UAA, UAG, UGA) is

reached

Initiation of Translation

• The initiation stage of translation brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome

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Elongation of the Polypeptide Chain

• In the elongation stage, amino acids are added one by one

to the preceding amino acid

Termination of Translation

• When the ribosome reaches a STOP codon, there is no corresponding transfer RNA

 a small protein called a “release factor” attaches to the stop codon.

 causes the whole complex to fall apart: messenger RNA, the two ribosome subunits, the new polypeptide.

• The messenger RNA can be translated many times, to produce many protein copies

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Polyribosomes

• A number of ribosomes can translate a single mRNA

molecule simultaneously forming a polyribosome

• Polyribosomes enable a cell to make many copies of a

polypeptide very quickly

Comparing Gene Expression In Prokaryotes And Eukaryotes

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Post-translation

• The new polypeptide is now floating loose in the cytoplasm if translated by a free ribosome

• Polypeptides fold spontaneously into their active

configuration, and they spontaneously join with other

polypeptides to form the final proteins

• Often translation is not sufficient to make a functional

protein, polypeptide chains are modified after translation

• Sometimes other molecules are also attached to the

polypeptides: sugars, lipids, phosphates, etc All of these

have special purposes for protein function

Targeting Polypeptides to Specific Locations

• Completed proteins are targeted to specific sites in the cell

• Two populations of ribosomes are evident in cells: free

ribsomes (in the cytosol) and bound ribosomes (attached

to the ER) (endoplasmic reticulum)

– Free ribosomes mostly synthesize proteins that function in the

cytosol

– Bound ribosomes make proteins of the endomembrane system

and proteins that are secreted from the cell

• Ribosomes are identical and can switch from free to bound

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Targeting Polypeptides to Specific Locations

• Polypeptide synthesis always begins in the cytosol

• Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach

it (polypeptide) to the ER

• Polypeptides destined for the ER or for secretion are marked by a signal peptide

• A signal-recognition particle (SRP) binds to the signal peptide

• The SRP brings the signal peptide and its ribosome to the ER

Ribosomes

mRNA Signal peptide Signal-

recognition

particle

(SRP)

SRP receptor protein CYTOSOL

ER LUMEN Translocation

complex

Signal peptide removed

ER membrane Protein

Mutation Causes and Rate

• The natural replication of DNA produces occasional errors DNA polymerase has an editing mechanism that

decreases the rate, but it still exists

• Typically genes incur base substitutions about once in

every 10,000 to 1,000,000 cells

• Since we have about 6 billion bases of DNA in each cell,

virtually every cell in your body contains several mutations

• Mutations can be harmful, lethal, helpful, silent

• However, most mutations are neutral: have no effect

• Only mutations in cells that become sperm or eggs—are

passed on to future generations

• Mutations in other body cells only cause trouble when

they cause cancer or related diseases

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Mutagens

• Mutagens are chemical or physical agents that interact with

DNA to cause mutations

• Physical agents include high-energy radiation like X-rays and ultraviolet light

• Chemical mutagens fall into several categories

– Chemicals that are base analogues that may be substituted into DNA,

they pair incorrectly during DNA replication.

– Interference with DNA replication by inserting into DNA and distorting

the double helix.

– Chemical changes in bases that change their pairing properties.

• Tests are often used as a preliminary screen of chemicals to

identify those that may cause cancer

• Most carcinogens are mutagenic and most mutagens are

carcinogenic