RNA and Protein Synthesis

RNA & Protein Synthesis

Uracil

Hydrogen bonds

Adenine

RiboseRNA

Basic components of RNA

Ribonucleic acid consists of following basic components

1- Ribose sugar 2- Phosphate in

diester linkage 4-Nitrogenous base

pairs- Purines- adenine,

guanine Pyrimidines- cytocine,

uracil

Primary structure of RNA

Although there are multiple types of RNA molecules, the basic structure of all RNA is similar.

Each kind of RNA is a polymeric molecule made by stringing together individual ribonucleotides, always by adding the 5'-phosphate group of one nucleotide onto the 3'-hydroxyl group of the previous nucleotide.

Secondary structure of RNA

Single-stranded RNA can also form many secondary structures in which a single RNA molecule folds over and forms hairpin loops, stabilized by intramolecular hydrogen bonds between complementary bases.

Such base-pairing of RNA is critical for many RNA functions, such as the ability of tRNA to bind to the correct sequence of mRNA during translation

DNA can replicate or undergo transcription

Replication-it is process by which DNA copies itself to produce identical daughter molecules of DNA. DNA is the reserve bank of genetic information.

Transcription-transcription results in the formation of one single-stranded RNA molecule.

DNA RNA

Structure Double Stranded

Single Stranded

Bases- Purines Adenine (A) Adenine (A)

Guanine (G) Guanine (G)

Bases - Pyrimidines

Cytosine (C) Cytosine (C)

Thymine (T) Uracil (U)

Sugar Deoxyribose Ribose

Differences between DNA and RNA:

RNA’s JOB= Make Proteins!!

Types of RNA

1) messenger RNA (mRNA)- carries instructions from the DNA in the nucleus to the ribosome

Types of RNA

2) ribosomal RNA (rRNA)- combines with proteins to form the ribosome (proteins made here)

3) transfer RNA (tRNA)- transfers each amino acid to the ribosome as it is specified by coded messages in mRNA during the construction of a protein

Types of RNA

4 ) snRNA – small nuclear RNA

5) snoRNA- small nucleolar RNA

6) scRNA- small cytoplasmic RNA

7) micro- RNAs,miRNA, small interfering RNAs

Present in eukaryotes only.

Protein Synthesis Overview

There are two steps to making proteins (protein synthesis):

1) Transcription (nucleus)

DNA RNA

2) Translation (cytoplasm)

RNA protein

DNA

Transcription

RNA

Translation

Protein

Conventional concept

Genome

Transcription

Transcriptome

Translation

Proteome

Current concept, Bioinformatics era

Proteins.

Everything a cell is or does depends on the proteins it contains.

From genes to proteins.

Two steps – Transcription , Translation.

Transcription

RNADNA

RNApolymerase

Adenine (DNA and RNA)Cytosine (DNA and RNA)Guanine(DNA and RNA)Thymine (DNA only)Uracil (RNA only)

Nucleus

TRANSCRIPTION

It is a process by which RNA is synthesize from DNA. The genetic information stored in DNA is expressed through RNA.

One of the two strands of DNA serves as Template and produces working copies of RNA molecules. The other DNA strand which does not participate in in transcription is referred to as coding strand or sense strand or non-template strand.

Transcription RNA Editing: Before the mRNA leaves the

nucleus, it is called pre-mRNA or (hnRNA) heterogeneous nuclear RNA and it gets “edited.” Parts of the pre-mRNA that are not involved in coding for proteins are called introns and are cut out. The remaining mRNA pieces are called exons (because they are expressed) and are spliced back together to form the mRNA.

Then the final mRNA leaves the nucleus through the nuclear pores and enters the cytoplasm headed to the ribosome.

Transcription 1) Transcription begins when the

enzyme RNA polymerase binds to DNA at a promoter region.

Promoters are signals in DNA that indicate to the enzyme where to bind to make RNA.

2) The enzyme separates the DNA strands by breaking the hydrogen bonds, and then uses one strand of DNA as a template from which nucleotides are assembled into a strand of RNA.

Transcription

3) RNA polymerase pairs up free floating RNA nucleotides with DNA template and joins the nucleotides together to form the backbone of the new mRNA strand.

4) When mRNA hits a termination sequence, it separates from the DNA

Steps of transcription

Initiation

Elongation

Termination

post – transcriptional modifications

The RNAs produced during transcription are called primary mRNA transcripts. They undergo many alterations- terminal base additions, base modifications, splicing etc. This process is required to convert RNA into active form. Enzyme involved mainly is - ribonucleases.

Cell

Nucleus

Cell

Nucleus

Nucleus

Chromosome

Key

= Phosphate

= Sugar

= Uracil

= Adenine

= Guanine

= Cytosine

RNA Polymerase

3’ 5’

Key

= Phosphate

= Sugar

= Uracil

= Adenine

= Guanine

= Cytosine

RNA Polymerase

3’ 5’

Key

= Phosphate

= Sugar

= Uracil

= Adenine

= Guanine

= Cytosine

RNA Polymerase

3’ 5’

Key

= Phosphate

= Sugar

= Uracil

= Adenine

= Guanine

= Cytosine

RNA Polymerase =

3’ 5’

Key

= Phosphate

= Sugar

= Uracil

= Adenine

= Guanine

= Cytosine

RNA Polymerase =

3’ 5’

Key

= Phosphate

= Sugar

= Uracil

= Adenine

= Guanine

= Cytosine

RNA Polymerase =

3’ 5’

mRNA Strand =

Key

= Phosphate

= Sugar

= Uracil

= Adenine

= Guanine

= Cytosine

RNA Polymerase =mRNA Strand =

3’ 5’

On average rate of RNA synthesis is about 43 nucleotides per second .

TRANSCRIPTION-COMPLIMENTARY BASE PAIR RELATIONSHIP

DNA 5’ A T G C A T G G C A 3’ CODING STRAND

3’ T A C G T A C C G T 5’ TEMPLATE STRAND

RNA 5’ …....A U G C A U G G C A………3’

The conventional numbering system of promoters

Bases preceding this are numbered

in a negative direction

There is no base numbered 0

Bases to the right are numbered in a

positive direction

Most of the promoter region is labeled with negative numbers

Promoter sites

In eukaryotes promoter DNA bases sequences known as HOGNESS BOX or TATA BOX located on the left about 25 nucleotides away(upstream) from the starting site of mRNA synthesis. Second site of recognition between 70 to 80 nucleotides upstream known as CAAT BOX.

Coding strand 5’ GGCCAATC ATATAA 3’

Template strand 3’ 5’

-70 bases -25 bases (coding region)

Start of transcription

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic promoter sequences are more variable and often much more complex than those of bacteria

For structural genes, at least three features are found in most promoters Regulatory elements TATA box (present in ~20 % of our genes) and other

short sequences in TATA-promoters that have a similar function

Transcriptional start site

Sequences of Eukaryotic Structural Genes

Factors that control gene expression can be divided into two types, based on their “location”

cis-acting elements DNA sequences that exert their effect only over a

particular gene Example: TATA box

trans-acting elements Regulatory proteins that bind to such DNA sequences

Sequences of Eukaryotic Structural Genes

Signals the end of protein synthesis

Usually an

adenine

The core promoter is relatively short It consists of the TATA box

Important in determining the precise start point for transcription

The core promoter by itself produces a low level of transcription This is termed basal transcription

Regulatory elements affect the binding of RNA polymerase to the promoter They are of two types

Enhancers Stimulate transcription

Silencers Inhibit transcription

They vary widely in their locations, from –50 to –100 region

RNA polymerases

RNA polymerase I- synthesis of precursors of large ribosomal RNAs.

RNA polymerases II- synthesizes the precursors for mRNAs and small rRNAs.

RNA polymerases III- formation of tRNAs and small rRNAs.


Three categories of proteins are required for basal transcription to occur at the promoter

RNA polymerase II six different proteins called general transcription factors

(GTFs or TFs) . They are- TFIID, TFIIA,TFIIB,TFIIF,TFIIE, TFIIH.

A protein complex called mediator.

RNA Polymerase II and its Transcription Factors


A closed complex

Released after the

open complex is formed

RNA poly II can now proceed to the elongation

stage

12-23Figure 12.7

12-26

Similar to the synthesis of DNA

via DNA polymerase

Figure 12.8

On average, the rate of RNA synthesis is about 43 nucleotides per second!

‘promoter’ Protein coding

Difference in gene structure between

- prokaryote

- eukaryotecore

‘promoter’

An important difference between prokaryotes and eukaryotes is that eukaryotes’ genes are not split into intons and exons. In eukaryotes is the DNA coding protein are, Therefore, exons eventually end up in the mRNA

intron

exons

Pre-mRNA

Transcription start, elongation, termination and RNA processing in eukaryotes

: coding protein: non-coding protein: ‘leader’ and ‘trailer’

CAP

CAP (poly A tail)

The longest gene in human genome is more than 1.500.000 base pares (bp) and the mRNA is ~ 7000 nt.

‘promoter’

intron

exons

GENE

mRNA AAAAAAAAAAAAAAn

TERMINATION Transcription stops by termination signals. Two

types of termination identified.

Rho depended- specific protein Rho factor, binds to the growing RNA, acts as ATPase and terminates transcription and releases RNA.

Rho independent – formation of hairpins of newly synthesized RNA.this occurs due to presence of palindromes. It is word that reads alike forward and backwards, like madam, motor. Presence of palindromes in DNA base sequence work as termination zone. Newly synthesize RNA folds to form hairpins due to complimentary base pairing, and termination occurs.

coding sequences, called exons, are interrupted by intervening sequences or introns

Transcription produces the entire gene product Introns are later removed or excised Exons are connected together or spliced

This phenomenon is termed RNA splicing It is a common genetic phenomenon in eukaryotes Occurs occasionally in bacteria as well

post transcriptional RNA modification

Aside from splicing, RNA transcripts can be modified in several ways

For example

Trimming of rRNA and tRNA transcripts

5’ Capping and 3’ polyA tailing of mRNA transcripts

y

RNA MODIFICATION

RNA Editing

Introns are removed and extrons are spliced

The spliceosome is a large complex that splices pre-mRNA

It is composed of several subunits known as snRNPs (pronounced “snurps”) Each snRNP contains small nuclear RNA and a set of

proteins. Or small nuclear ribonucloprotein particle. Types of snRNPs are U1,U2,U3,U4,U5,U6.

Pre-mRNA Splicing

The subunits of a spliceosome carry out several functions

1. Bind to an intron sequence and precisely recognize the intron-exon boundaries

2. Hold the pre-mRNA in the correct configuration

3. Catalyze the chemical reactions that remove introns and covalently link exons

Pre-mRNA Splicing

Intron loops out and exons brought closer

together

Intron will be degraded and the snRNPs used again

One benefit of genes with introns is a phenomenon called alternative splicing

A pre-mRNA with multiple introns can be spliced in different ways This will generate mature mRNAs with different

combinations of exons

This variation in splicing can occur in different cell types or during different stages of development

Intron Advantage?

The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single gene

This allows an organism to carry fewer genes in its genome

Intron Advantage?

Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’ end This event is known as capping

Capping occurs as the pre-mRNA is being synthesized by RNA pol II Usually when the transcript is only 20 to 25 bases long

Capping: marking 5’ends of mRNAs

The 7-methylguanosine cap structure is recognized by cap-binding proteins

Cap-binding proteins play roles in the

Movement of some RNAs into the cytoplasm Early stages of translation Splicing of introns

Function of Capping

Most mature mRNAs have a string of adenine nucleotides at their 3’ ends This is termed the polyA tail

The polyA tail is not encoded in the gene sequence It is added enzymatically after the gene is completely

transcribed

The 3’ end of a mRNA: Tailing

Cell

Nucleus

Cell

Nucleus

RNAPolymerase

RNA Polymerase binds and unwinds the DNA double helix.


RNAPolymerase

Guanine

Cytosine

Thymine

Adenine


RNAPolymerase

Guanine

Cytosine

Thymine

Adenine

RNA Polymerase binds and unwinds the DNA

double helix.

RNAPolymerase

Guanine

Cytosine

Thymine

Adenine

RNA Polymerase binds and unwinds the DNA double

helix.

RNAPolymerase

Guanine

Cytosine

Thymine

Adenine


helix.

RNAPolymerase


helix.

RNAPolymerase

RNA Polymerase binds to the promoter region.

RNAPolymerase


RNAPolymerase


RNAPolymerase


RNAPolymerase


RNAPolymerase


RNAPolymerase

Guanine

Cytosine

Thymine

Adenine

RNA Polymerase reads the DNA and creates the mRNA strand.

RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil

Start Codon Coding Region


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand


RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil


mRNA Strand

mRNA leaves the nucleus and enters the cytoplasm.

RNAPolymerase

Guanine

Cytosine

Thymine

AdenineUracil

Start Codon Stop CodonCoding Region

mRNA Strand

Termination Sequence



Nuclear Pore


Nuclear Pore


Nuclear Pore


Nuclear Pore


Nuclear Pore


Nuclear Pore


The Genetic CodeProteins (polypeptides) are long chains of amino acids that

are joined together.

There are 20 different amino acids.

The structure and function of proteins are determined by the order in which different amino acids are joined together to produce them.

The four bases (letters) of mRNA (A, U, G, and C) are read three letters at a time (and translated) to determine the order in which amino acids are added to a protein.

AMINO ACIDS

Amino acids are organic solvents.

Have two functional groups –NH₂ and

-COOH group.

The amino group is basic while carboxylic group is acidic in nature.

Soluble in water but insoluble in organic solvents e.g. chloroform,acetone,ether,etc.

All amino acids which make up proteins are L-α-aminoacids.

Semi-essential aminoacids.

These include Arginine and Histidine.These are growth promoting factors since they are not synthesized in sufficient quantity during growth.

SELENOCYSTEINE- the 21st amino acid.

The Genetic Code

A codon consists of three consecutive nucleotides that specify a single amino acid that is to be added to the polypeptide (protein).

The Codon Table

Sixty-four combinations are possible when a sequence of three bases are used; thus, 64 different mRNA codons are in the genetic code.

Some codons do not code for amino acids; they provide instructions for making the protein.

More than one codon can code for the same amino acid.

All organisms use the same genetic code (A,T,C,G). This provides evidence that all

life on Earth evolved from a common origin.

Cracking the Code This picture shows the amino

acid to which each of the 64 possible codons corresponds.

To decode a codon, start at the middle of the circle and move outward.

Ex: CGA

Arginine

Ex: GAU

Aspartic Acid

Translation

Translation takes place on ribosomes, in the cytoplasm.

The cell uses information from messenger RNA (mRNA) to produce proteins, by decoding the mRNA message into a polypeptide chain (protein).

Stapes of protein synthesis

1) requirements of the components- amino acids, ribosome, mRNA,tRNA, ATP

2)activation of amino acids

3)protein synthesis proper

4) chaperones and protein folding

5) post – translational modifications.

Source: http://www.coolschool.ca/lor/BI12/unit6/U06L01.htm

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Cytoplasm

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Start Codon Codon Codon Codon Stop Codon

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Key

= Uracil

= Adenine

= Guanine

= Cytosine


Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine


Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Codon Codon Codon Stop Codon

Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Codon Codon Stop Codon

Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Codon Codon Stop Codon

Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Codon Stop Codon

Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Codon Stop Codon

Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Amino Acid Chain

Key

= Uracil

= Adenine

= Guanine

= Cytosine

Ribosome

Anticodon

Amino AcidtRNA

Polypeptide Chain

Peptide Bond

Final Protein in Tertiary Structure

translation

Initiation codon

AUG

Termination codons or non-sense codons or stop signals

UAA

UAG

UGA

Messenger RNA (mRNA)

1) The mRNA that was transcribed from DNA during transcription, leaves the cell’s nucleus and enters the cytoplasm.

Transfer RNA(tRNA) 2) The mRNA enters the cytoplasm and attaches to a ribosome at the

AUG, which is the start codon. This begins translation.

3) The transfer RNA (tRNA) bonds with the correct amino acid and becomes “charged.” (in the cytoplasm)

4) The tRNA carries the amino acid to the ribosome. Each tRNA has an anticodon whose bases are complementary to a

codon on the mRNA strand. (The tRNA brings the correct amino acid to the ribosome.)

Ex: The ribosome positions the start codon to attract its anticodon, which is part of the tRNA that binds methionine.

The ribosome also binds the next codon and its anticodon.

The Polypeptide “Assembly Line”

5) The ribosome moves along the mRNA and adds more amino acids to the growing polypeptide or protein

The tRNA floats away, allowing the ribosome to bind to another tRNA.

The ribosome moves along the mRNA, attaching new tRNA molecules and amino acids.

Completing the Polypeptide6) The process continues

until the ribosome reaches one of the three stop codons on the mRNA, and then the ribosome falls off the mRNA.

7) The result is a polypeptide chain or protein that is ready for use in the cell.

mRNA binds to the ribosome and the code is read.

mRNA binds to the ribosome and the code is read.

tRNA has the anticodon and amino acid attaches.

Guanine

Cytosine Adenine

Uracil

tRNA

anticod

on

amino

acid

Met

start

codon

Amino acids bind to each other through peptide bonds.

Guanine

Cytosine Adenine

Uracil

tRNA

anticod

on

amino

acid

peptide bondLeuArg

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticod

on

amino

acid

peptide bondLeuArgLeu

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticod

on

amino

acid

peptide bondLeuArgLeuThr

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticod

on

amino

acid

peptide bondLeuArgLeuThrGlu

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticod

on

amino

acid

peptide bondLeuArgLeuThrGluThr

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticod

on

amino

acid

peptide bondLeuArgLeuThrGluThrAsp

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticod

on

amino

acid

peptide bondLeuArgLeuThrGluThrAspCys

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticodon

amino

acid

peptide bondLeuArgLeuThrGluThrAspCysLeu

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticodon

amino

acid

peptide bondLeuArgLeuThrGluThrAspCysLeuThr

start

codon


Guanine

Cytosine Adenine

Uracil

tRNA

anticodon

amino

acid

peptide bondLeuArgLeuThrGluThrAspCysLeuThrSTOP

start

codon

Ribosome hits the stop codon, and protein synthesis is complete.

Guanine

Cytosine Adenine

Uracil

amino

acid

chain

peptide bondLeuArgLeuThrGluThrAspCysLeuThrAsp

Stop Codon

CompletedProtein

start

codon


Guanine

Cytosine Adenine

Uracil

amino

acid

chain


Stop Codon

CompletedProtein


Guanine

Cytosine Adenine

Uracil

amino

acid

chain


Stop Codon

CompletedProtein

Amino acid chain coils into a complete protein.

CompletedProtein

Amino acid chain coils into a complete protein.

CompletedProtein

Source: http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture1/Lecture1.html

Technology

RNA and Protein Synthesis