Composicin de las protenas

Composicin, sntesis, estructura y funcin de las protenas

Grupo amino Grupo cido carboxlico Carbn- Cadena variable

Peptide bond formation between amino acids

amino acids H R1 side chain C NH2 COOH H side chain

H2N

C R2 COOH

Aminocidos

Peptidil transferasa

H2O
H

R1 C CONH C R2

N-terminus

NH2

COOH

C-terminus

1 BASE Un aminocido 2 BASES Un aminocido 3 BASES Un aminocido

41 = 4 42 = 16 43 = 64

X X

cido asprtico (Asp), cido glutmico (Glu), Alanina (Ala), Arginina (Arg), Asparagina (Asn), Cisteina (Cys), Fenilalanina (Phe), Glicina (Gly), Glutamina (Gln), Histidina (His), Isoleucina (Ile), Leucina (Leu), Lisina (Lys), Metionina (Met), Prolina (Pro), Serina (Ser), Tirosina (Tyr), Treonina (Thr), Triptfano (Trp), Valina (Val).

64 tripletos posibles (codon) solo 20 aminocidos (1966)

The genetic code is almost universal, but there are a few exceptions / variations: Eukaryotic Mitochondria (human) UGA = Trp (not stop) AUA = Met (not Ile) AGA Stop (not Arg) AGG Changes in nuclear codes of certain ciliated protozoans (a class of euks) AGA AGG =STOP - strong selective pressure to maintain the genetic code in its current form

UAA, UAG, UGA = CODONES DE TERMINACION

AUG = CODON DE INICIACION

Sntesis de las protenas - traduccin

Prokaryotic vs. Eukaryotic Translation
- general processes are very similar - eukaryotic ribosomes are larger. 4.2 MDa vs 2.7 MDa - in Eukaryotes, special MetinitiatortRNA is NOT formylated - IMPORTANT: no euk. Shine-Delgarno sequence. Initiation occurs at the first start codon after 5-cap - additional initiation and Elongation Factors required in eukaryotes

Principales elementos implicados - eucariotas

1. ARNm maduro (instrucciones). 2. Amino cidos 3. Factores de iniciacin eucariotas (eukariotic Initiation Factors): eIF1, eIF2, eIF3, eIF4, eIF5, eIF6. 4. Guanosil trifosfato (GPT). 5. Ribosomas (40S y 60s). 6. Factores que promueven el reconocimiento directo del codn de iniciacin (ITAFs: IRES trans-acting factors). 7. ARNt cargados con cada uno de los 20 aminocidos (traductores). La asociacin ARNt + aminocido es catalizada por las aminoacil-ARNt sintetasas, especifica para cada par ARNt-aminocido (p.e. metionil-ARNt sintetasa, glutaminil-ARNt sintetasa, etc.) 8. Factor de elongacin (eEF1). 9. Complejo de factores de liberadores (eRFs+GTP).

ARNm
AUG UAA UAG UGA

A triplet code has 3 potential READING FRAMES

Reading frames
5 3 a b c d e f ATGTTTGCTGACGGTTTAACGGAAGGCGGAAACATGGCGAAGAAAAAACCAGTAGAAAAA ---------+---------+---------+---------+---------+---------+ TACAAACGACTGCCAAATTGCCTTCCGCCTTTGTACCGCTTCTTTTTTGGTCATCTTTTT M F A D G L T E G G N M A K K K P V E K C L L T V * R K A E T W R R K N Q * K K V C * R F N G R R K H G E E K T S R K K ---------+---------+---------+---------+---------+---------+ H K S V T * R F A S V H R L F F W Y F F N A S P K V S P P F M A F F F G T S F T Q Q R N L P L R F C P S S F V L L F 3 5

--CAGAAUAUCAUCUAUUCUACA--

1.

asn ile ile tyr ser thr 2. ile ser ser ile leu xxx 3. tyr his leu phe tyr xxx

6 reading frames (af) in dsDNA (of course, only 3 in mRNA) ORF = Open Reading Frame From start codon to stop codon

3 totally different potential sequences

Open Reading Frames (ORFs)

long (>100 codons i.e. 300 bases, 100 amino acids) continuous stretches of DNA sequence between a START and a STOP codon

ORFs have the potential to encode proteins

AAATTGTAAA CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTTAA AT CAGCTCAT TTTTTAACCA ATAGGCCGAA ATCGGCAAAA TCCCTTATAA ATCAAAAGAA TAGACCGAGA TAGGGTTGAG TGTTGTTCCA GTTTGGAACA AGAGTCCACT ATTAAAGAAC GTGGACTCCA ACGTCAAAGG GCGAAAAACC GTCTATCAGG GCGATGGCCC ACTACGTGAA CCATCACCCT AATCAAGTTT TTTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT AAAGGGAGCC CCCGATTTAG AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA GGGAAGAAAG CGAAAGGAGC GGGCGCTAGGGCGCTGGCAA GTGTAGCGGT CACGCTGCGC GTAACCACCA CACCCGCCGC GCTTAATGCG CCGCTACAGG GCGCGTCCCA TTCGCCATTC AGGCTACGCA ACTGTTGGGA AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAGGGGG GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA AAACGACGGC CAGTGAATTG TAATACGACT CACTATAGGG CGAATTGGAG CTCCACCGCG GTGGCGGCCG CTCTAGAACT AGTGGATCCC CCGGGCTGCA GGAATTCGAT ATCAAGCTTA TCGATACCGT CGACCTCGAG GGGGGGCCCG GTACCCAGCT TTTGTTCCCT TTAGTGAGGG TTAATTCCGA GCTTGGCGTA ATCATGGTCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT AGGAGCCGGA AGCATAAAGT GTAAAGCCTG GGGTGCCTAA TGAGTGAGGT AACTCACATT AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AGCTGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCG TAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CGGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTCCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCAATGCTCA CGCTGTAGGT ATGTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTGACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTG CCCGTCGTGT

Expressed sequence tags: expression profiles in different tissues

Expressed sequence tags (ESTs) : Partially sequenced cDNA clone. single-pass sequenced cDNAs from an mRNA population derived from a specified cell population (eg. a specific tissue, organ, developmental state or environmental condition). cover only a part of the total mRNA sequence depending on the method of their synthesis, there are 3' or 5' ESTs primary applications are to provides a profile of the mRNA population, and to serve as a quick method for cloning a large number of genes known to be expressed in a cell population. GenBank contains several million human EST sequences from different tissue libraries.

Analysis of ORFs allows predictions of proteins from genomic DNA sequence

Common features
1. All single nucleotide chains, 73-93 ribonucleotides

ARNt

2. Contain many unusual bases 3. The 5 end is always phosphorylated and usually a pG 4. The 3 end is always a -CCA-OH 5. Extensive base pairing, highly conserved 3-D structure
pseudo uridine

6. Anticodon loop contains 7 bases

Inosine: a modified base also found in tRNA

NH2
guanine has NH2 group

An unusual property of inosine (I) is that it can base pair with A,C or U - this makes it very useful in many experimental techniques (e.g., degenerate PCR where one is uncertain about the precise sequence the primer should have at certain locations)

Variability between certain domains of different tRNAs

Amnoacil ARNt sintetasas

3 2

Existen 20 Amnoacil ARNt sintetasas

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Alanyl-tRNA synthetase Arginyl-tRNA synthetase Asparaginyl-tRNA synthetase Aspartyl-tRNA synthetase Cysteinyl-tRNA synthetase Glutaminyl-tRNA synthetase Glutamyl-tRNA synthetase Glycyl-tRNA synthetase Histidyl-tRNA synthetase Isoleucyl-tRNA synthetase Leucyl-tRNA synthetase Lysyl-tRNA synthetase Methionyl-tRNA synthetase Phenylalanyl-tRNA synthetase Prolyl-tRNA synthetase Seryl-tRNA synthetase Threonyl-tRNA synthetase Tryptophanyl-tRNA synthetase Tyrosyl-tRNA synthetase Valyl-tRNA synthetase

1. La enzima tiene que seleccionar el AA correcto 2. La enzima tiene que escoger el ARNt correspondiente

Identity Elements in tRNA

- we might predict that the tRNA synthetases would only use the anticodon to determine the tRNA identity (bind the correct tRNA)

Sometimes one tRNA will recognize more than one codon

- what the enzyme actually does is examine other unique structural features that identify the tRNA - the location of some of these are indicated in the secondary structures shown in the figure

Wobble
61 codons for amino acids, but many cells have less than 61 tRNAs Some tRNAs can recognize several codons (for same amino acid) Third position in codon (mRNA) and first position in anticodon (tRNA) can have less stringent (non-Watson-Crick) base-pairing

6. Prokaryotic ribosome an RNA/protein complex consisting of 2 subunits

large

Ribosomas

50S 70S 30S

small

50S 23S rRNA 5S rRNA

32 proteins

30S 16S rRNA

21 proteins

S = Sedimentation coefficient a

combined measure of size and shape

Eukaryotic ribosome 2 subunits

large

60S 80S 40S

small

60S 28S rRNA 5.8S rRNA 5S rRNA

50 proteins

40S 18S rRNA

33 proteins

Las tres fases de la traduccin:

1.Iniciacin 2.Elongacin 3.Terminacin

Iniciacin - procariotas
Most proteins begin on AUG (GUG & UUG less frequently) The first amino acid incorporated (in prokaryotes) is Methionine.

1. Iniciacin

- Methionine of an initiator tRNA is modified: Formylated

O R (amino acid side chain) HC-NH-CH-COOH

- Note: f-Met looks like a peptide! - normal tRNAmet only recognizes AUG while tRNAf-Met will recognize AUG, GUG & UUG codons - f-Met only used at initiation

How does translation start? AUG is the Start Codon

How does ribosome distinguish start AUG and internal AUG? Shine-Dalgarno Sequence in prokaryotic and phage mRNAs

How is the Shine-Dalgarno Sequence Recognized? 30S ribosomal subunit

Abot 5-10 nucleotides upstream of AUG start codon. Recognized by base pairing to 16S rRNA (small subunit)

Iniciacin - eucariotas
3 key proteins involved, these are known as Initiation Factors (IFs)
IF 1 & 3 aid in the disassociation of the 30S & 50S subunits. IF 2 - a GTPase which presents the initiator tRNA
metionil-ARNt

Elongacin - procariotas

Elongacin

Elongacin - eucariotas

Terminacin
A

Terminacin - procariotas

Procariotas

- termination requires Release Factors (RFs) & GTP

Terminacin - eucariotas

Polisomas en eucariotas y procariotas

Complejo de factores liberadores (eRF1, GTP, eRF3)

Polisomas circulares en eucariotas

Estructura de las protenas

(b) Estructura secundaria Nivel de estructura Primaria Secundaria Estructura Fuerzas de estabilizacin (a)Estructura primaria

Secuencia linear de aminocidos La secuencia de amino cidos adquiere conformaciones en forma de espiral, llamadas hlices-, o de hojas plegadas, llamadas hojas-. Las cadenas secundarias se repliegan en el espacio por medio de interacciones entre cadenas laterales (p.e.: protenas globulares, protenas fibrilares) Varios polipptidos con estructura terciaria se combinan para formar grandes agregados (p.e. la Hemoglobina est formada por 4 unidades)

Enlaces covalentes (enlaces peptdicos). Enlaces de H.

Grupo heme Puentes salinos. Fuerzas de Vander Wals. Enlaces de H. Uniones disulfuros (-S-S-), etc. Puentes salinos Fuerzas Vander Wals. Enlaces de H. Uniones disulfuro (-S-S-), etc. (c) Estructura terciaria (c) Estructura cuaternaria

Terciaria

Cuaternaria

Funcion de la proteinas