PROTEIN SYNTHESIS & REGULATION OF GENE EXPRESSION

Abdul Salam M. Sofro Faculty of Medicine & LPPM YARSI University Jakarta RC Biotechnology UGM Yogyakarta

Teaching aims
By the end of the lecture: students are expected to understand the molecular or biochemical processes of DNA replication, transcription and protein synthesis Students are expected to understand the principles of gene expression

Core topics
Overview DNA Structure & replication of DNA Transcription of DNA (RNA synthesis) Protein synthesis (translation) Regulation of gene expression

Overview
Protein biosynthesis is also called translation (translation of information from four-letter language & structure of nucleic acid into 20letter language & structure of protein) This process requires: Informational mRNA exported from nucleus bilingual tRNA that reads the message Ribosomes that serve as catalytic & organizational centers A variety of protein factors & energy

Cont.
Polypeptide/proteins are formed by sequential addition of amino acids in the specific order determined by info carried in the nucleotide sequence of the mRNA Proteins are often matured or processed by a variety of modifications Levels of translation is regulated

 Cells vary in their need & ability to synthesized proteins: Growing cells & dividing cells must synthesize much larger amounts of protein Some cells synthesize proteins for export as well as for their own use (e.g. liver cells synthesize large numbers of enzymes needed for their metabolic pathways as well as proteins for export including serum albumin)

 Terminally differentiated (adult) red blood cells have no nuclei, do not divide & do not synthesize proteins due to the absence of components of the biosynthetic apparatus

 Components of the translational apparatus mRNA Ribosomes tRNA

mRNA
Carrier of information present in DNA In eukaryotes (including human) usually are synthesized as larger precursor molecules that are processed prior to export from the nucleus It has several identifying characteristics: almost always monocistronic (encoding a single polypeptide)

 5 end is capped with 7-methylG followed by non-translated region which may be short or up to a few hundred nucleotides in length separated the cap with translational initiation signal (AUG) Uninterrupted sequences that specify a unique polypeptide sequence; followed by 3-untranslated sequence, usually about 100 nucleotides in length, before terminated by a 100-200 nucleotide long poly-A tail

 In prokaryotes: 5 terminus is not capped Mostly polycistronic (encoding several polypeptides & include more than one initiation AUG sequence) Ribosome positioning sequence is located about 10 nucleotides upstream of a valid AUG initiation signal An untranslated sequence follows the termination signal, but no poly-A tail

Ribosome
Workbenches for polypeptide/protein biosynthesis Have two dissimilar subunits, each contains RNA & many proteins

tRNA
A bilingual translator molecule All tRNA molecules have several common structural characteristics (3-terminal CCA sequence to bind amino acid, a highly conserved cloverleaf secondary structure & L-shape three dimensional structure) Great specificity in interaction with mRNA & the aminoacyl-tRNA synthetase

Transfer of genetic information

Replication of DNA transmission of genetic information from parental cell to its daughter cells Transcription of DNA transmission of genetic information from DNA to RNA Translation of RNA (polypeptide/protein biosynthesis) transmission of genetic information from RNA to polypeptide/protein

DNA structure & Replication of DNA

DNA structure & Replication of DNA

DNA is a macromolecule that ultimately controls every aspect of cellular functions through protein synthesis DNA is a transforming agent as well as material responsible for transmitting genetic information from one generation to the next

DNA

RNA

Protein

Human Genome Size

NUCLEAR GENOME * 23 pairs of chromosomes 2 x ( 3 x 109 b.p) 2 meters DNA / Cell * 2 x ( 3 x 1012 cells) meters DNA in human body 8,000 x earth to moon

MITOCHONDRIAL GENOME * circular, 16,569 bp

DNA (deoxyribonucleic acid)

Sugar is deoxyribose DNA is a polymer of deoxyribonucleotides Bases are adenine (A), guanine (G), cytosine (C) and thymine (T) Double strands anti parallel

Replication of DNA
Takes place in nucleus Both strands act as template (35 strand) Originated from replication fork or replication bubble Factors involved: Helicase DNA binding proteins DNA polymerase Primase dNTP (dATP, dGTP, dCTP, TTP) & many others

 Needs RNA primer Produces : Leading strand of new DNA (complementary to old DNA template with free 3-OH end) Lagging strand of new DNA with Okazaki fragments (complementary to old DNA template with free 5- end

Image Source: http://esg-www.mit.edu:8001/esgbio/dogma/repl.html

Image Source: http://esg-www.mit.edu:8001/esgbio/dogma/repl.html

Transcription of DNA (RNA synthesis)

RNA (ribonucleic acid)

Sugar is ribose RNA is a polymer of ribonucleotides. Bases are adenine, guanine, cytosine and uracil (instead of thymine) Single strand Three types of RNA: mRNA, tRNA & rRNA

Transcription of DNA (RNA synthesis)

In chromosomes, DNA acts as a template for the synthesis of RNA in a process called transcription: Only one strand of DNA act as template (35 strand) Originated from any point of DNA of the gene (Polypeptide gene, tRNA gene or rRNA gene) at the promotor site Does not require RNA primer

 Involved: RNA polymerase NTP (ATP, GTP, CTP, UTP) Termination signal In most mammalian cells, only 1% of the DNA sequence is copied into a functional RNA (mRNA). Only one part of the DNA is transcribed to produce nuclear RNA, and only a minor portion of the nuclear RNA survives the RNA processing steps.

Promoter
Bind RNA polymerase protect DNA from digestion Two common motifs on 5 : -10 sequence 5-TATAAT-3 and -35 sequence (6 bp long) 5-TTGACA-3 At coding strand = sense (+) strand & template strand = antisense (-) strand

 Strong vs. weak promoter (every 2 sec & once in 10 min.) Specific sequences near it influenced by regulatory proteins & interact with RNA polymerase Recognized by sigma subunit RNA polymerase (2 holoenzyme)

RNA polymerase
Searches DNA for initiation site There are many more molecules of RNA polymerase per cell than DNA polymerase. RNA polymerase proceeds at a rate much slower than DNA polymerase (approximately 50-100 bases/sec for RNA versus near 1000 bases/sec for DNA the fidelity of RNA polymerization is much lower than DNA

 Unwinds a short stretch of double-helical DNA to produce DNA template Select correct dNTP & catalyses formation of fosfodiester bond Interact with activator & repressor protein that modulate the rate of transcription Unwinds nearly two turns of template DNA before initiating RNA synthesis Starts with pppG or pppA Primers are not needed

DNA template
Transcription bubble for elongation containing RNA pol, DNA, nascent RNA Form RNA-DNA hybrid helix (about 12 bp long/one turn of A-DNA) Direct RNA synthesis Transcribed by RNA pol (lack nuclease activity) with lower fidelity than that of replication (error rate 1 in 104 or 105)

Nascent RNA & processing

Undergo little or no modification for mRNA (maybe translated while being transcribed) Cleaved & modified for rRNA & tRNA in E coli, a primary RNA transcript is excised to generate three rRNAs (5S, 16S & 23S) & one tRNA by ribonuclease P May contain arrays of several kinds of tRNAs or several copies of same tRNA Addition of nucleotides to termini of some RNA chains (CCA to 3 tRNA) Modifications of bases & ribose units of rRNAs

Transcription termination
Formation of fosfodiester bonds ceases RNA-DNA hybrid dissociates Melted DNA region rewinds RNA pol releases DNA Precisely controlled Stop signals in DNA template regions e.g. palindromic GC-rich region followed by AT-rich region forms RNA hairpin structure Rho protein helps terminate transcription

One of the most important stages in RNA processing is RNA splicing. In many genes, the DNA sequence coding for proteins, or "exons", may be interrupted by stretches of noncoding DNA, called "introns".

In the cell nucleus, the DNA that includes all the exons and introns of the gene is first transcribed into a complementary RNA copy called "nuclear RNA," or nRNA.

In a second step, introns are removed from nRNA by a process called RNA splicing. The edited sequence is called "messenger RNA," or mRNA.

Protein synthesis (Translation of mRNA)

Translation of RNA
The ribosome binds to the mRNA at the start codon (AUG) that is recognized only by the initiator tRNA. The ribosome proceeds to the elongation phase of protein synthesis. During this stage, complexes, composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon.

 The ribosome moves from codon to codon along the mRNA. Amino acids are added one by one, translated into polypeptidic sequences dictated by DNA and represented by mRNA. At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome.

Codon
Three-letter code words ( a triplet code) Unambiguous Non-overlapping Without punctuation Universal Can be found either in DNA (sense strand) and mRNA

The collection of codons (64) makes up the genetic code

Three nonsense codons (UAA, UAG, UGA) do not code for specific amino acid and are utilized as termination signal

A = adenine G = guanine C = cytosine T = thymine U = uracil DNA transfers information to mRNA in the form of a code defined by a sequence of nucleotides bases. During protein synthesis, ribosomes move along the mRNA molecule and "read" its sequence three nucleotides at a time (codon) from the 5' end to the 3' end.

 Each amino acid is specified by the mRNA's codon, and then pairs with a sequence of three complementary nucleotides carried by a particular tRNA (anticodon). Since RNA is constructed from four types of nucleotides, there are 64 possible triplet sequences or codons (4x4x4).

 Three of these possible codons specify the termination of the polypeptide chain. They are called "stop codons (nonsense codons). That leaves 61 codons to specify only 20 different amino acids. Therefore, most of the amino acids are represented by more than one codon. The genetic code is said to be degenerate.

Amino acids specified by each codon sequence on mRNA

Cys: Cysteine Asp: Aspartic Glu: Glutamic acid acid

Ala: Alanine

Phe: Phenylalanine Gly: Glycine

Ile: His: Histidine Isoleucine

Asn: Asparagine

Lys: Lysine

Met: Leu: Leucine Methionine Gln: Glutamine Val: Valine

Pro: Proline
Thr: Threonine

Arg: Arginine
Trp: Tryptophane

Ser: Serine
Tyr: Tyrosisne

Protein translation takes place by the following steps

1. Formation of the initiation complex 2. Elongation of the polypeptide chain (one repetition of the steps a, b and c for every amino acid incorporated into the protein being made): a. binding of aminoacyl-tRNA b. peptide bond formation c. translocation 3. Termination

Remember !
Proteins are polypeptides made up of individual amino acids linked together, Carbohydrates are polysaccharides made up of individual monosaccharides linked together, and Nucleic acids are polynucleotides made up of individual nucleotides linked together.

Mutations
Result when changes occur in the nucleotide sequence may not occur in the template strand but appear after replication Some mutations occur by base substitution single base changes (point mutations): Transitions (pryrimidine to other pyrimidine, purine to other purine) Transversion (pyrimidine to purine or purine to pyrimidine)

 Single base changes in DNA sequence followed by changes in mRNA molecules may have one of several effects when translated into protein: No detectable effect silent mutation Missense effect missense mutation Appearance of nonsense codon that result in premature termination of polypeptide chain being synthesized nonsense mutation

 Substitution of amino acids in protein causes missense mutations (illustration on Hemoglobin molecule):
Acceptable missense mutations Hb Hikari: AAA or AAG (lys) to AAU or AAC (asp) Hb E: GAA or GAG (glu) to AAA or AAG (lys) Partially acceptable missense mutations Hb S: GAA or GAG (glu) to GUA or GUG (val) Unacceptable missense mutations Hb M: Hb (Fe2+) to met Hb (Fe3+)

 Frameshift mutations result from deletion or insertion of nucleotides generates altered mRNAs May be one, two, three or multiples nucleotides

Regulation of gene expression

 In bacteria & viruses: Alteration of gene expression is required by organism to adapt to environmental changes involves interaction of specific binding proteins with various regions of DNA in the immediate vicinity of the transcription start site

 In eukaryotes: in addition to those proteins, alteration of gene expression also involves tissue specific expression; regulation by hormones, metals & chemicals; gene amplification; gene rearrangement; posttranscriptional modification

Type of responses to a regulatory signal Type A: increased rate of gene expression that is dependent upon the continued presence of inducing signal Type B: increased rate of gene expression that transient even in the continued present of regulatory signal Type C: increased rate of gene expression that persist indefinitely even after the termination of the signal

Type of gene regulation

Positive regulation: The expression of genetic info is quantitatively increased by the presence of a specific regulatory element (the molecule is positive regulator) Negative regulation: the expression of genetic info is diminished by the presence of a specific regulatory element (the molecule is negative regulator)

Model of gene expression in prokaryotes

One cistron, one subunit concept instead of one gene one enzyme concept (cistron is the smallest unit of gene expression, coding for the structure of the subunit of a protein molecule) Inducible genes: their expression increases in response to an inducer Constitutive genes: their expression is reasonably constant (not known to be subject to regulation)

The earliest level of regulation is at DNA level during transcription

Legend: Process of creating a hybrid strand of DNA/RNA The two strands of a DNA molecule are denatured by heating to about 100C = 212F (a to b). At this temperature, the complementary base pairs that hold the double helix strands together are disrupted and the helix rapidly dissociates into two single strands. The DNA denaturation is reversible by keeping the two single stands of DNA for a prolonged period at 65C = 149F (b to a). This process is called DNA renaturation or hybridization. Similar hybridization reactions can occur between any single stranded nucleic acid chain: DNA/DNA, RNA/RNA, DNA/RNA. If an RNA transcript is introduced during the renaturation process, the RNA competes with the coding DNA strand and forms double-stranded DNA/RNA hybrid molecule (c to d). These hybridization reactions can be used to detect and characterize nucleotide sequences using a particular nucleotide sequence as a probe.